Il-10 variant molecules and methods for treating inflammatory disease and oncology

ABSTRACT

The application relates to compositions or formulations comprising variant IL-10 molecules, fusion proteins, and chimeric proteins thereof useful for the treatment of cancer, inflammatory diseases or disorders, and autoimmune diseases or disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/814,669, filed Mar. 6, 2019, U.S. Provisional Patent ApplicationNo. 62/899,504, filed Sep. 12, 2019, and U.S. Provisional PatentApplication No. 62/962,332, filed Jan. 17, 2020, the disclosures of eachare herein incorporated by reference in their entireties.

INTRODUCTION

The application relates to variant forms of Interleukin 10 (IL-10) thatinclude modifications to the IL-10 receptor binding region and/or thedomains responsible for the inter-domain angles that exists in the IL-10molecule. By modifying one or both of these domains in IL-10, theinventors have surprisingly found that the resulting biological functionof the IL-10 receptor may be tuned or modulated to elicit a specificbiological response. The application also relates to a half-lifeextended IL-10 or IL-10 variant molecule that include non-protein basedserum extension moieties as well as protein based extension modalities.The Application also relates to fusion proteins comprising the IL-10variant molecules.

BACKGROUND

IL-10 has been described as cytokine synthesis inhibitory factor, due toits capacity to inhibit both (i) pro-inflammatory cytokine secretion bymonocytes/macrophages in response to lipopolysaccharide, and (ii)interleukin 2 (IL-2) secretion and proliferation of CD4⁺ T cells. Whenviral analogues of IL-10 were discovered and reported to share similaror identical functions to human IL-10, it was presumed that these viralanalogs of IL-10enhanced viral virulence by adopting the function of asuppressive cytokine found in the human genome.

Further investigation of the suppressive effects of IL-10 was madepossible by the generation of the IL-10 knockout mice that developchronic enterocolitis. The data generated from these mice clearlyillustrated that IL-10 knockout mice develop severe inflammationthroughout the gastrointestinal track, predominantly through chronicinflammatory cytokine secretion by monocytes/macrophages and CD4⁺ Tcells, which is consistent with initial in vitro observations.

Collectively the data implied that IL-10 exerted a dominant role insuppressing inflammation. In particular, patients lacking functionalIL-10 receptors, or the ability to produce IL-10 exhibited an increasedpredisposition to developing inflammation associated diseases of thegastrointestinal track.

Multiple clinical trials were conducted to evaluate theanti-inflammatory function of IL-10 in context of psoriasis, rheumatoidarthritis, and Crohn's disease. In general, recombinant human IL-10(rHuIL-10) treatment was found to be safe but lacking in efficacy. Inparticular, treating Crohn's patients with rHuIL-10 lead to an inversedose response, where low doses appeared to moderately inhibitinflammation and the suppressive effect was lost at high doses. Rigorousanalysis of the final Crohn's study revealed that patients dosed with 10and 20 μg/kg rHuIL-10 exhibited increased serum concentrations ofinterferon gamma (IFNγ) and Neopterin. IFNγ is known to worsen thepathogenesis of inflammatory bowel disease, and Crohn's disease. Thedata suggests that at high doses, treatment with IL-10 induces IFNγ,which, in turn, will exacerbate inflammatory disease.

Further analysis of IL-10 effects in healthy humans suggests thatadministration of IL-10 before exposure to the pro-inflammatory factorlipopolysaccharide (LPS) inhibits production of pro-inflammatorycytokines. However, administering rHuIL-10 after exposure to LPSenhanced the secretion of pro-inflammatory cytokines. Since LPS is aproduct of both normal and foreign gut bacteria in patients withInflammatory Bowel Disease (IBD), these patients will never be “free” ofLPS and therefore will never be in a state where IL-10 treatment couldbe applied prior to LPS. Thus, these data suggests that Crohn's andpatients with other inflammatory diseases will never see the therapeuticbenefits of IL-10 treatment.

Adding further confusion to the role of IL-10 is the accumulating dataindicating that IL-10 activates the immune system to induce anti-tumorresponses. Initial data suggested that IL-10 activated NK cells, butfurther investigation uncovered that IL-10 treatment inhibits tumorgrowth in a CD8⁺ T cell and IFNγ dependent manner. Continuedinvestigation revealed that IL-10 treatment inhibits FoxP3⁺CD4⁺ Tregulatory cell proliferation, and enhanced Kupffer cell scavenging,which are all functions suggesting that IL-10 is a potent immunestimulant, rather than a suppressor. Lastly, these stimulatoryactivities where confirmed in clinical studies of oncology patientstreated with PEGylated IL-10.

Collectively, these data indicates that IL-10 treatment of patientssuffering from autoimmune associated inflammation failed to elicit atherapeutic benefit, suggesting that IL-10 is not a pan-immunesuppressant. In keeping with this, treating cancer patients with (PEG)IL-10 lead to potent and therapeutically useful immune activation,specifically the induction of dose dependent serum IFNγ, similar toCrohn's patients treated with IL-10, implying that IL-10 is a potentimmune stimulant.

Unresolved then is why viruses would acquire the IL-10 sequence. Furtheranalysis of both the Epstein Barr Virus (EBV-IL10) and Cytomegalovirus(CMV-IL10) homologs suggest these viruses have altered the native IL-10sequence in two predominant ways. The EBV-IL10 appears to retain asimilar tertiary angle of homodimer interaction leading to a specificangle of ligand receptor interaction, while substantially decreasingit's affinity for the IL-10 receptor. The CMV-IL10 exhibits an increasedaffinity for the IL-10 receptor while substantially altering the angleof interaction with the IL-10 receptors. While the EBV-IL10 and CMV-IL10sequences respectively retain approximately 80% and 27% homology tonative IL-10, each of the viral homologs have completely different IL-10receptor affinities, different angles of receptor engagement, with eachviral homologues appearing to exert highly similar anti-inflammatoryfunctions to native IL-10. It is therefore unclear how the affinity forthe IL-10 receptors and/or the angle of receptor interaction affects thesubsequent downstream transduction of the IL-10 signal.

SUMMARY OF VARIOUS PREFERRED EMBODIMENTS

The present application generally relates to novel IL-10 variantmolecules that modulate IL-10 receptor signal transduction. Thus, theapplication relates to IL-10 variant molecules that incorporatemodifications to the structure of an IL-10 molecule, resulting in novelIL-10 variant molecules having altered IL-10 receptor binding affinitiesand/or altered IL-10 inter-domain angles. The inventor surprisinglydiscovered that modifying IL-10 in key domains impacting IL-10 receptoraffinity and/or IL-10 inter-domain angles resulted in the creation ofIL-10 receptor agonists that may be used in treating immune diseases,inflammatory diseases or conditions, as well as in treating cancer.Moreover, the IL-10 variant molecules may also have increased serumhalf-life by incorporating the variant IL-10 molecules as part of afusion protein, such as various antibody domains (Fc or variabledomains), without impeding the monomers of IL-10 or the IL-10 variantsfrom forming an IL-10 homodimer.

In certain embodiments, the IL-10 variant molecule is modified humanIL-10. In other embodiments, the IL-10 variant molecule is modifiedmouse IL-10. In preferred embodiments, the IL-10 variant molecule is amodified viral homolog of IL-10. In a more preferred embodiment, theviral IL-10 homolog is CMV-IL10. In a most preferred embodiment, theviral IL-10 homolog is EBV-IL10.

In further embodiments, the IL-10 variant molecule incorporates at leastone or more amino acid additions, deletions or substitutions within thereceptor binding region. In yet other embodiments, the IL-10 variantmolecule incorporates at least one or more amino acid additions,deletions or substitutions in a region responsible for forming theinter-domain angle of IL-10. In a preferred embodiment, the IL-10variant molecule incorporates one or both of the modifications to thereceptor binding domain and/or the region responsible for theinter-domain angle. In a most preferred embodiment, the IL-10 variantmolecule incorporates one or both modifications in an EBV-IL10 proteinmolecule.

In various embodiments, the IL-10 variant molecule, when compared towild-type IL-10, has enhanced affinity to the IL-10 receptor or receptorcomplex. In other embodiments, the IL-10 variant molecule, when comparedto the wild-type IL-10, has diminished affinity to the IL-10 receptor orreceptor complex. In other embodiments, the IL-10 variant molecule formsa narrower or constrained inter-domain angle, when compared to thewild-type IL-10, more preferably EBV-IL10. In another embodiment, theIL-10 variant molecule forms a wider or relaxed inter-domain angle, whencompared to the wild-type IL-10, more preferably EBV IL-10.

In yet further embodiments, the EBV IL-10 variant molecules incorporateat least one or more amino acid additions, deletions or substitutionswithin the receptor binding region located in the helix A, the AB loop,and/or the helix F (“site 1”) of EBV IL-10. The modifications to thereceptor binding domain region may, in certain embodiments, increase orenhance the affinity to the IL-10 receptor or receptor complex. Incertain other embodiments, modifications to the receptor binding regionmay diminish or decrease the affinity to the IL-10 receptor or receptorcomplex

In further embodiments, the EBV IL-10 variant molecules incorporate atleast one or more amino acid additions, deletions or substitutionswithin the EBV IL-10 regions responsible for inter-domain angleformation. In preferred embodiments, the modifications may occur in theDE loop of EBV IL-10. The modifications in the DE loop result in EBVIL-10 variant molecules that have a constrained inter-domain angle or arelaxed inter-domain angle.

In other aspects, the IL-10 variant molecules are chimeric or fusionmolecules. In one embodiment, the chimeric or fusion protein willcomprise one or more domains from different proteins or mutations withina single protein giving the characteristics of another protein. In apreferred embodiment, the chimeric or fused protein will include a firstportion comprising an IL-10 variant molecule as described herein fusedto another molecule including, but not limited to albumin, enzymes,glycosyltransferases, galactosyltransferases, IgG hinge regions (such asan Fc region), one or more variable domains (such as but not limited toa variable heavy chain or a variable light chain) of one or moreantibodies, cytokines (such as IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7,IL-8, IL-9, IL-12, IL-15, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β,and tumor necrosis factors -α, -β), labels, agents, chemotherapeuticagents, radioisotopes, and half-life extenders (such as hydroxyl ethylstarch (HESylation), polysialic acids, heparosan polymers, elastin-likepolypeptides and hyaluronic acid). In still another embodiment, theIL-10 variant molecule is fused to one or more antibody heavy or lightchain variable regions. The fusion proteins may include one or morelinkers that covalently linked the different parts of the fusionprotein. The fusion protein may form a non-covalently bound complex withanother fusion protein of the same type. Such a fusion protein willallow monomers of IL-10 or monomers of the variant IL-10 molecule toassociate together into a functional homodimer of IL-10 or variantIL-10.

In other embodiments, the variant IL-10 molecule is part of anengineered fusion protein. The fusion protein will comprise at least onemonomer of an IL-10 or an IL-10 variant molecule conjugated at a firstterminal end of the fusion protein to a linker or spacer, wherein theone or more spacers are used to link the various parts of the fusionprotein, which is then conjugated to at least one other moleculeconjugated at the other terminal end, wherein the molecule is selectedfrom at least one cytokine or monomer thereof, a therapeutic agent, alabel, a serum half-life extension molecule, or a protein (such as butnot limited to a receptor, ligand, or various portions of an antibody).In a preferred embodiment, the fusion protein comprises a monomer ofIL-10 or a monomer of an IL-10 variant molecule conjugated, via linkersor spacers, to at least one heavy and/or light chain variable region. Ina most preferred embodiment, the fusion protein comprises a monomer ofEBV IL-10 or a monomer of an EBV IL-10 variant molecule conjugated, vialinkers or spacers, to at least one heavy and/or light chain region. Ina most preferred embodiment, a monomer of EBV IL-10 or EBV IL-10 variantmolecule thereof is conjugated to one heavy chain variable region andone light chain variable region, where the monomers together form adimer complex. The monomer of EBV IL-10 or monomer of EBV IL-10 variantmolecule maybe conjugated to either the amino terminal or carboxyterminal end of a heavy or light chain variable region.

In certain embodiments, a therapeutic amount of the IL-10 variantmolecule, fusion protein or chimeric protein thereof of the applicationis administered to a subject suffering from an inflammatory disease orcondition, such as but not limited to inflammatory bowel disease (IBD),Crohn's disease, Ulcerative colitis, nonalcoholic steatohepatitis(NASH), and nonalcoholic fatty liver disease (NAFLD). In anotherembodiment, a therapeutic amount of the IL-10 variant molecule, fusionprotein or chimeric protein thereof of the application is administeredto a subject suffering from cancer. Treatment of subjects having morethan one pathological condition is also envisioned. In a more preferredembodiment, the IL-10 variant molecule is an EBV-IL10 variant, fusionprotein or chimeric protein thereof. In yet another embodiment, theIL-10 variant molecule, fusion protein or chimeric protein thereof isused in combination therapy. For example, the IL-10 variant molecule,fusion protein or chimeric protein thereof may be administered to asubject in conjunction with other therapies or treatments. In yet otherembodiments, a therapeutic amount of the IL-10 variant molecule, fusionprotein or chimeric protein thereof of the application is administeredto a subject suffering from lipid based diseases, such as but notlimited to elevated levels cholesterol.

In various embodiments, the IL-10 variant molecule or fusion proteinthereof of the present application is delivered as an isolated andpurified protein. The delivery may be in the form of a subcutaneousbolus injection or in the form of multiple microinjections into thedermis and subcutaneous tissue. In yet another embodiment, the IL-10variant molecule may be delivered as a nucleic acid vector comprising asequence encoding the IL-10 variant, fusion protein or chimeric proteinthereof. The nucleic acid vector can be a plasmid or a viral particlecarrying a viral vector. In one embodiment, the IL-10 variant moleculesmay be delivered to a subject by genetic medicine techniques generallyknown to those of skill in the art.

In other embodiments, the IL-10 variant molecule may also beadministered as part of a combination therapy regimen. In oneembodiment, the IL-10 variant molecule can be administered incombination with Bispecific T-Cell Engagers (BITES), immunotherapiescurrently available for the treatment of cancer, IBD, Crohn's disease,NAFLD, NASH, and autoimmune diseases, for example.

In another embodiment, the nucleic acid vector is an adeno-associatedvirus(AAV) vector having one or more AAV inverted terminal repeat (ITR)sequence elements and control elements for directing expression of thesequence encoding the IL-10 variant molecule in a target cell, which AAVvector can be administered either as a plasmid (“naked” DNA) or aspackaged in an AAV particle. In another embodiment, the nucleic acidvector is a vaccinia virus vector. A variety of vaccinia viral vectorsderived from different strains may be used to introduce the variantIL-10 molecules of the present application, such as WR strain (ATCCVR-119), the Wyeth strain (ATCC VR-325), the Lederle-Chorioallantoicstrain (ATCC VR-325), the CL strain (ATCC VR-117), and others; all ofthese strains are available from the American Type Culture Collection(Manassas, Va.).

In another embodiment, any of the IL-10 variant molecules describedherein, include but not limited to PEGylated IL-10 variant molecules,may be delivered to a subject by any method described herein orgenerally known in the art.

These and other embodiments of the subject application will readilyoccur to those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a ribbon diagram (Josephson et al, Immunity, 15, p. 35-46)of a monomeric IL-10 molecule. Portions highlighted in red indicatessite I receptor contact interface and green indicates site II receptorcontact interface.

FIGS. 2A-2C compare the ability of IL-10 and EBV IL-10 to activatemonocytes/macrophages (Mθ) by examining IL-1β (FIG. 2A) and TNFα (FIG.2B) cytokine production and the stimulation of T-cells by examining IFNγ(FIG. 2C) production in Donor 1.

FIGS. 3A-3C compare the ability of IL-10 and EBV IL-10 to activatemonocytes/macrophages (Mθ) by examining IL-1β (FIG. 3A) and TNFα (FIG.3B) cytokine production and the stimulation of T-cells by examining IFNγ(FIG. 3C) production in Donor 1.

FIGS. 4A-B show the amount of IFNγ induction from T cells followingstimulation by IL-10 or EBV IL-10.

FIGS. 5A-5C show the effect of N-terminal 5 kDa mono and di-PEGylatedEBV-IL-10 on MC/9 cell proliferation, (FIG. 5A), TNFα secretion bymonocytes/macrophages (Mθ) in response to LPS (FIG. 5B), and IFNγsecretion by T cells in response to T cell receptor stimulation (FIG.5C).

FIGS. 6A-6E show specific amino acid sequences for various IL-10 variantmolecules and fusion proteins comprising EBV-IL-10.

FIG. 7 shows a schematic representation of a fusion protein comprisingan IL-10 or IL-10 variant molecule conjugated to a linker or spacer (inthis case a scFv). This representative example shows IL-10 or IL-10variant molecules conjugated for both the N-terminus and the C-terminus.

FIGS. 8A-8C are schematic representations of various IL-10 variants,such as variants made in EBV IL-10. FIG. 8A (DV05) is an IL-10 variantcomprising a single point mutation at amino acid position 31 (V31L V31L)of SEQ ID No. 3. FIG. 8B (DV06) is an IL-10 variant comprising a singlepoint mutation at amino acid position 75 (A75I A75I) of SEQ ID No. 3.FIG. 8C (DV07) is an IL-10 variant comprising a two point mutations atamino acid positions 31 and 75 (V31L and A75I) of SEQ ID No. 3.

FIG. 8D assays monocytes/macrophage response to various forms of IL-10,including wild type human IL-10, EBV-IL-10, DV05, DV06, and DV07. Allforms, including the IL-10 variants—DV05, DV06, DV07—suppress TNFαsecretion in response to LPS.

FIG. 8E assays T-cell response to various forms of IL-10—wild type humanIL-10, EBV IL-10, DV05, DV06, and DV07—and not all forms induceIFN-gamma.

FIGS. 9A-9F are a schematic representation of various configurations ofIL-10 fusion protein/immunoconjugate/diabody constructs. FIGS. 9(a)-(c)represent a fusion protein complex (i.e., a diabody) where each fusionprotein comprises a VH and VL regions obtained from two differentantibodies linked to a monomer of IL-10 (which may also be substitutedwith an IL-10 variant molecule) via a carboxy terminal linker or anamino terminal linker with (a) a single mutation—e.g., amino acidposition 31—impacting IL-10 receptor binding; (b) a single mutation—e.g.amino acid position 75—impacting IL-10 receptor binding; and (c) twomutations—e.g. amino acid positions 31 and 75—impacting IL-10 receptorbinding. FIGS. 9(d)-(f) represent a fusion protein complex (i.e., aminibody) where each fusion protein comprises a single VH or VL regionobtained from one antibody linked to a monomer of IL-10 (which may alsobe substituted with an IL-10 variant molecule) via a carboxy terminallinker or an amino terminal linker with (d) a single mutation—e.g.,amino acid position 31—impacting IL-10 receptor binding; (e) a singlemutation—e.g., amino acid position 75—impacting IL-10 receptor binding;and (f) two mutations—e.g., amino acid positions 31 and 75—impactingIL-10 receptor binding.

FIGS. 10(A)-10(F) are a schematic representations of variousconfigurations of the IL-10 fusion protein/immunoconjugate/diabodyconstructs. FIGS. 10(a)-(c) represents a single fusion protein (i.e.,minibody) where the monomers of IL-10 (which may also be substitutedwith an IL-10 variant molecule) are each linked via a carboxy terminallinker or an amino terminal linker to either a VH or VL from the sameantibody and the VH and VL are linked together. The monomers of IL-10comprise (a) a single mutation—e.g., amino acid position 31—impactingIL-10 receptor binding; (b) a single mutation—e.g., amino acid position75—impacting IL-10 receptor binding; and (c) two mutations—e.g., aminoacid positions 31 and 75—impacting IL-10 receptor binding. FIGS.10(d)-(f) represents a single fusion protein where monomers of IL-10(which may also be substituted with an IL-10 variant molecule) arelinked together and each monomer of IL-10 is further linked via acarboxy terminal linker or an amino terminal linker to a single VH or VLregion obtained from one antibody. The IL-10 monomers comprise (d) asingle mutation—e.g., amino acid position 31) impacting IL-10 receptorbinding; (e) a single mutation—e.g., amino acid position 75—impactingIL-10 receptor binding; and (f) two mutations—e.g., amino acid positions31 and 75) impacting IL-10 receptor binding.

FIG. 11 shows the in vivo reduction of tumor volume using a diabodycomprising the DV07 mutation. Formulation buffer (1× Phosphate BufferedSaline; “control”) and a DV07 diabody (a fusion protein complexcomprising the EBV IL-10 variant with V31L V31L and A75I A75I of themature protein and variable domains from anti-CD3α and anti-EGFR,neither of these VH/VL pais bind to the mouse target) were administeredat a single dose at day 3. The dose concentrations of 1 mg/kg and 0.4mg/kg were tested for the DV07 diabody.

FIG. 12 shows the in vitro T-cell response to a published variant ofIL-10 (diamond), wild-type IL-10 (circle) and an alternative form of aDV07 diabody termed DHivDEbo:DV07 (diamond; a fusion protein complexcomprising the EBV IL-10 variant with V31L and A75I mutations andvariable domains from anti-HIV and anti-Ebola (neither of these VH/VLpairs bind to mouse proteins), where the diabody has a structureschematically represented by FIG. 9(c)).

FIG. 13A compares the suppression of TNFα induced by LPS exposure tomonocytes/macrophages using various forms of the EBV IL-10 variantmolecule with the V31L and A75I mutations in diabody form. “wt”represents human IL-10; “EBV” is EBV IL-10; “DCd3DEgfr:DV05” is adiabody comprising VH and VL regions from an anti-CD3 antibody and ananti-EGFR antibody linked to an EBV IL-10 variant comprising a V31Lmutation; “DCd3DEgfr:DV07” is a diabody comprising VH and VL regionsfrom an anti-CD3 antibody and an anti-EGFR antibody linked to an EBVIL-10 variant comprising both V31L and A75I mutations; “DHivDEbo:DV07”is a diabody comprising VH and VL regions from an anti-HIV antibody andan anti-Ebola antibody linked to an EBV IL-10 variant comprising bothV31L and A75I mutations.

FIG. 13B compares the secretion of IFNγ in T-cell in response assayusing human IL-10 (“wt”) EBV IL-10 (“EBV”) to various diabody formscomprising an EBV IL-10 variant molecules with the V31L and A75Imutations and VH and VL regions from different antibodies. FormDHivDEgfr:DV07 is an EBV IL-10 variant diabody with V31L and A75Isubstitutions comprising variable regions from anti-HIV and anti-EGFR.Form DHivDEbo:DV07 is an EBV IL-10 variant diabody with both V31L andA75I mutations comprising variable regions from anti-HIV and anti-Ebola.

FIGS. 14A-14B compare two forms of the EBV IL-10 variant diabody,DH:DV07 and DHDE:DV07 on monocytes/macrophages (FIG. 14A) and T-cells(FIG. 14B) isolated from two donors. Form DHDV07 is an EBV IL-10 variantdiabody with V31L and A75I substitutions comprising variable regionsfrom anti-HIV and anti-EGFR. Form DHDE:DV07 is an EBV IL-10 variantdiabody with V31L and A75I substitutions comprising variable regionsfrom anti-HIV and anti-Ebola. FIG. 14A compares the suppression of TNFαinduced by LPS in isolated monocytes/macrophages using human IL-10(“wt”), DH:DV07 and DHDE:DV07. FIG. 14B compares the secretion of IFNγin isolated T-cell in response to human IL-10 (“wt”), EBV IL-10, DH:DV07and DHDE:DV07.

FIG. 15 is a direct comparison of various forms of EBV-10 variantdiabody forms on MC/9 mast cells. This assay compares human IL-10, EBVIL-10, D:DV05 (EBV IL-10 with a V31L mutation comprising variableregions from anti-CD3α and anti-EGFR), D:DV06 (EBV IL-10 with a A75Imutation comprising variable regions from anti-CD3α and anti-EGFR),D:DV07 with V31L and A75I mutations comprising variable regions fromanti-CD3α and anti-EGFR), and DhivDEbo:DV07 (EBV IL-10 variant diabodywith V31L and A75I substitutions comprising variable regions fromanti-HIV and anti-Ebola).

FIGS. 16A-16C are results from an in vivo tumor study using D:DV07 (withV31L and A75I mutations comprising variable regions from anti-CD3α andanti-EGFR). In vivo tumor volume were assessed following theadministration of a dosing formulation buffer (“control”), 0.4 mg/kgthree times a week (q3w), 0.2 mg/kg three times a week (q3w), 0.2 mg/kgtwo days off (qd), 0.1 mg/kg two days off (qd).

FIGS. 17A-17B are results from an in vivo studies of two IL-10 variantfusion protein formats, large molecular weight (large) and smallmolecular weight (small), that correspond to the schematic diagrams ofFIGS. 9C and 9F respectively. The IL-10 variant comprises V31L and A75Imutations. These results tested the impact of IL-10 fusion proteinswithout targeting capabilities to reduce tumor size. The VH and VLregions from the fusion proteins are non-targeting sequences, fromeither an anti-HIV and anti-ebola (large) or anti-ebola (small). FIG.17A is a dosing study of the non-targeting small format with a dosing of5 days on, 2 days off, as compared pegylated IL-10 (0.75 mg/kg daily).FIG. 17B is a dosing study of the non-targeting small (1 mg/kg, 0.5mg/kg, 0.25 mg/kg) and large (0.2 mg/kg) formats with dosing three timesa week compared to pegylated recombinant human IL-10 (0.75 mg/kg daily).

FIGS. 18A-18C are results from an in vivo study of two IL-10 variantfusion protein formats, large and small, that correspond to theschematic diagrams of FIGS. 9C and 9F, respectively. These resultstested the impact of IL-10 fusion proteins with targeting capabilitiesto reduce tumor size. The IL-10 variant comprises V31L and A75Imutations. In the large format, one set of the VH and VL region from thefusion proteins are from an anti-EGFR antibody, while the other set ofthe VH and VL is from an anti-ebola antibody. The small format includeVH and VL from just an anti-EGFR antibody. FIG. 18A are the results froma daily dosing study of targeted IL-10 fusion proteins, large format(0.25 mg/kg), small format (0.25 mg/kg), and non-targeted IL-10 fusionprotein small format (0.25 mg/kg) as compared to pegylated recombinanthuman IL-10 (0.75 mg/kg). FIG. 18B are the results from a three times aweek dosing study of large format targeted IL-10 fusion protein (1mg/kg, 0.25 mg/kg, and 0.25 mg/kg daily) as compared to large formatnon-targeted IL-10 fusion protein (DhDe:DV07 at 0.2 mg/kg) and pegylatedIL-10 (0.75 mg/kg daily). FIG. 18C are the results from a three times aweek dosing study of small format targeted IL-10 fusion protein (1mg/kg, 0.25 mg/kg) as compared to small format non-targeted IL-10 fusionprotein (Debo:DV07 at 1 mg/kg) and pegylated IL-10 (0.75 mg/kg daily)

FIGS. 19A-19B are results from an in vivo cholesterol study using adiabody with an EBV IL-10 variant with a V31L mutation comprisingvariable regions from anti-CD3α and anti-EGFR.

FIGS. 20A-20B are results from an in vitro comparative study on twoIL-10 variant fusion proteins on macrophage and T-cells. The assaysexamine the in vitro effectiveness of two forms of a DV06 fusion proteinin comparison to human IL-10. FIG. 20A is a monocyte/macrophage assayusing DhivDebo:DV06 (SEQ ID Nos: 26 and 27) and DmadcamDEbo:DV06 (SEQ IDNos: 41 and 42). FIG. 20B is a T-cell response assay as measured byIFN-gamma using DhivDebo:DV06 (SEQ ID Nos: 26 and 27) andDmadcamDEbo:DV06 (SEQ ID Nos: 41 and 42).

FIGS. 21A-21D are EBV IL-10 amino acid sequences. FIG. 21A is EBV IL-10.FIG. 21B is DV05 including a V31L substitution. FIG. 21C is a DV06including a A75I substitution. FIG. 21D is DV07 including both V31L andA75I substitutions.

DETAILED DESCRIPTION OF VARIOUS PREFERRED EMBODIMENTS

Before describing the various embodiments application in detail, it isto be understood that this application is not limited to particularformulations or process parameters as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing various embodiments only, and is not intended tobe limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the variousdescribed embodiments, the preferred materials and methods are describedherein.

Unless otherwise indicated, the embodiments described herein employconventional methods and techniques of molecular biology, biochemistry,pharmacology, chemistry, and immunology, well known to those skilled inthe art. Many of the general techniques for designing and fabricatingthe IL-10 variants, including but not limited to human, CMV and/or EBVforms of IL-10, as well as the assays for testing the IL-10 variants,are well known methods that are readily available and detailed in theart. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplaneds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell ScientificPublications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc.,current addition). N-terminal aldehyde based PEGylation chemistry isalso well known in the art.

The following terms will be used to describe the various embodimentsdiscussed herein, and are intended to be defined as indicated below.

As used herein in describing the various embodiments, the singular forms“a”, “an” and “the” include plural referents unless the content clearlydictates otherwise.

The term “about”, refers to a deviance of between 0.0001-5% from theindicated number or range of numbers. In one embodiment, the term“about”, refers to a deviance of between 1-10% from the indicated numberor range of numbers. In one embodiment, the term “about”, refers to adeviance of up to 25% from the indicated number or range of numbers. Ina more specific embodiment, the term “about” refers to a difference of1-25% in terms of nucleotide sequence homology or amino acid sequencehomology when compared to a wild-type sequence.

The term “interleukin-10” or “IL-10” refers to a protein comprising twosubunits non-covalently joined to form a homodimer, where IL-10 is anintercalated dimer of two six helix bundle (helix A-F). As used herein,unless otherwise indicated “interleukin-10” and “IL-10” can refer tohuman IL-10 (“hIL-10”; Genbank Accession No. NP_000563; or U.S. Pat. No.6,217,857) protein (SEQ ID No: 1) or nucleic acid (SEQ ID No: 2); mouseIL-10 (“mIL-10”; Genbank Accession No: M37897; or U.S. Pat. No.6,217,857) protein (SEQ ID No: 7) or nucleic acid (SEQ ID No: 8); orviral IL-10, (“vIL-10”). Viral IL-10 homologs may be derived from EBV orCMV (Genbank Accession Nos. NC_007605 and DQ367962, respectively). Theterm EBV-IL10 refers to the EBV homolog of IL-10 protein (SEQ ID No: 3)or the nucleic acid (SEQ ID No: 4). The term CMV-IL10 refers to the CMVhomolog of IL-10 protein (SEQ ID No: 5) or the nucleic acid (SEQ ID No:6). The term monomeric IL-10, as used herein, refers to the individualsubunits of IL-10 or variant IL-10 that, when non-covalently joined,form a homodimer of IL-10 or variant IL-10. The terms “wild-type,” “wt”and “native” are used interchangeably herein to refer to the sequence ofthe protein (e.g. IL-10, CMV-IL10 or EBV-IL10) as commonly found innature in the species of origin of the specific IL-10 in question. Forexample, the term “wild-type” or “native” EBV-IL10 would thus correspondto an amino acid sequence that is most commonly found in nature.

The term “derive,” “derived,” “derive from,” or “derived from,” is usedherein to identify the original source of a molecule, such as a viralform of IL-10 molecule, but is not meant to limit the method in whichthe molecule is prepare, manufactured, fabricated, or made. This wouldinclude methods, such as but not limited to, chemical or recombinantmeans.

The term “derivative” is intended to include any suitable modificationof the reference molecule of interest or of an analog thereof, such assulfation, acetylation, glycosylation, phosphorylation, polymerconjugation (such as with polyethylene glycol), hesylation, or otheraddition of foreign moieties, so long as the desired biological activity(e.g., anti-inflammation and/or no T cell stimulation) of the referencemolecule or the variant is retained.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule, that retain a desired activity,such as, for example, anti-inflammatory activity. Generally, the terms“variant,” “variants,” “analog” and “mutein” as it relates to apolypeptide refers to a compound or compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (that are conservative in nature), and/ordeletions, relative to the native molecule. As such, the terms “IL-10variant”, “variant IL-10,” “IL-10 variant molecule,” and grammaticalvariations and plural forms thereof are all intended to be equivalentterms that refer to an IL-10 amino acid (or nucleic acid) sequence thatdiffers from wild-type IL-10 anywhere from 1-25% in sequence identity orhomology. Thus, for example, an EBV IL-10 variant molecule is one thatdiffers from wild-type EBV IL-10 by having one or more amino acid (ornucleotide sequence encoding the amino acid) additions, substitutionsand/or deletions. Thus in one form, an EBV IL-10 variant is one thatdiffers from the wild type sequence of SEQ ID No.:3 by having about 1%to 25% difference in sequence homology, which amounts to about 1-42amino acid difference.

The term “fusion protein” refers to a combination or conjugation of twoor more proteins or polypeptides that results in a novel arrangement ofproteins that do not normally exist naturally. The fusion protein is aresult of covalent linkages of the two or more proteins or polypeptides.The two or more proteins that make up the fusion protein may be arrangedin any configuration from amino-terminal end to carboxy-terminal end.Thus for example, the carboxy-terminal end of one protein may becovalently linked to either the carboxy terminal end or the aminoterminal end of another protein. Exemplary fusion proteins may includecombining a monomeric IL-10 or monomeric variant IL-10 molecule with oneor more antibody variable domains. The fusion proteins may also formdimers or associated with other fusion proteins of the same type, whichresults in a fusion protein complex. The complexing of the fusionprotein may in some cases activate or increase the functionality of afusion protein when compared to a non-complexed fusion protein. Forexample, a monomeric IL-10 or monomeric variant IL-10 molecule with oneor more antibody variable domains may have limited or decreased capacityto bind to an IL-10 receptor; however, when the fusion protein iscomplexed, the monomeric forms of IL-10 or variant IL-10 molecule becomea homodimer and the variable domains associate into a functionaldiabody.

A “functional variant” is an IL-10 variant molecule that includesmodifications (e.g., additions, substitutions, and/or deletions) that donot destroy the biological activity of the reference molecule. Thesevariants may be “homologous” to the reference molecule as defined below.In general, the amino acid sequences of such analogs will have a highdegree of sequence homology to the reference sequence, e.g., amino acidsequence homology of more than 50%, generally more than 60%-70%, evenmore particularly 80%-85% or more, such as at least 90%-95% or more,when the two sequences are aligned. Often, the analogs will include thesame number of amino acids but will include substitutions. Thefunctional variant will retain biological activity that is enhanced,diminished or substantially the same as the native molecule.Specifically, the term “variant” IL-10 molecule, which isinterchangeable with the terms “engineered” IL-10 molecule or IL-10variant molecule or IL-10 variant, refers to an IL-10 molecule orprotein that includes one or both modifications to the IL-10 receptorbinding domain(s) and/or to the regions responsible for forming aninter-domain angle or inter-homodimeric angle in the IL-10 molecule orprotein. A variant IL-10 “fusion protein” or “diabody” or “fusion”generally refers to the formation of a fusion protein (or a fusionprotein complex) comprising variant IL-10 (in either monomeric form orin homodimeric form) and at least one other protein. As used herein avariant IL-10 “or a fusion protein thereof” may be used throughout thisdescription to describe such a variant IL-10 fusion protein.

An “analog” or “analogs” may include substitutions that are conservativein nature. For example, conservative substitutions might include in kindtype substitutions such as, but not limited to (1) an acidicsubstitution between aspartate and glutamate; (2) a basic substitutionbetween any one of lysine, arginine, or histidine; (3) a non-polarsubstitution between any one of alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, or tryptophan; and (4) a unchargedpolar substitution between any one of glycine, asparagine, glutamine,cysteine, serine threonine, or tyrosine. Phenylalanine, tryptophan, andtyrosine are sometimes classified as aromatic amino acids. It is alsopossible that an isolated replacement of leucine with isoleucine orvaline, an aspartate with a glutamate, a threonine with a serine, or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid may be made so long as the desired and specificbiological activity is intact. For example, the polypeptide of interestmay include up to about 1-10 conservative or non-conservative amino acidsubstitutions, or even up to about 15-25 conservative ornon-conservative amino acid substitutions, or any integer between 1-50,so long as the desired function of the molecule remains intact. One ofskill in the art may readily determine regions of the molecule ofinterest that can tolerate change well known in the art.

A “mutein” further includes polypeptides having one or more aminoacid-like molecules including but not limited to compounds comprisingonly amino and/or imino molecules, polypeptides containing one or moreanalogs of an amino acid (including, for example, unnatural amino acids,etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring (e.g., synthetic), cyclized, branched moleculesand the like. Preferably, the analog or mutein will retain somebiological activity that is enhanced, diminished or substantially thesame as the native molecule. Methods for making polypeptide analogs andmuteins are well known in the art.

The term “homolog,” “homology,” “homologous” or “substantiallyhomologous” refers to the percent identity between at least twopolynucleotide sequences or at least two polypeptide sequences.Sequences are homologous to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%-85%, preferably at least about 90%, and most preferably atleast about 95%-98% sequence identity over a defined length of themolecules.

The term “sequence identity” refers to an exact nucleotide-by-nucleotideor amino acid-by-amino acid correspondence. The sequence identity mayrange from 100% sequence identity to 50% sequence identity. A percentsequence identity can be determined using a variety of methods includingbut not limited to a direct comparison of the sequence informationbetween two molecules (the reference sequence and a sequence withunknown % identity to the reference sequence) by aligning the sequences,counting the exact number of matches between the two aligned sequences,dividing by the length of the reference sequence, and multiplying theresult by 100. Readily available computer programs can be used to aid inthe identification of percent identity.

The term “fragment” is intended to include a portion molecule of thefull-length amino acid or polynucleotide sequence and/or structure. Afragment of a polypeptide may include, for example, a C-terminaldeletion, an N-terminal deletion, and/or an internal deletion of thenative polypeptide. Active or functional fragments of a particularprotein will generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, or any integer between 5 amino acids andthe full-length sequence, provided that the fragment in question retainsbiological activity, such as anti-inflammatory activity. As it relatesto antibodies, an antibody fragment refers to a portion of an intactantibody comprising an antigen binding site or variable region (heavychain and/or light chain regions) of the intact antibody. These mayinclude, for example, the Fab, Fab′, Fab′-SH, (Fab′)₂, Fv fragments,diabodies, single chain Fv (ScFv), single chain polypeptides with onelight chain variable domain, a fragment having three CDRs of the lightchain variable domain or heavy chain variable domain.

The term “substantially purified” generally refers to isolation of asubstance such that the substance comprises the majority percent of thesample in which it resides. A substantially purified component comprises50%, preferably 80%-85%, more preferably 90-95% of the sample. Likewisethe term “isolated” is meant, when referring to a polypeptide or apolynucleotide, that the indicated molecule is separate and discretefrom the whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macro-moleculesof the same type.

The terms “subject,” “individual” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murine, rodent, simian, human, farm animals,sport animals, and certain pets.

The term “administering” includes routes of administration which allowthe active ingredient of the application to perform their intendedfunction.

A “therapeutically effective amount” as it relates to, for example,administering the EBV-IL-10 variants or fusion proteins thereofdescribed herein, refers to a sufficient amount of the EBV-IL-10 variantor fusion proteins thereof to promote certain biological activities.These might include, for example, suppression of myeloid cell function,enhanced Kupffer cell activity, and/or lack of any effect on CD8⁺ Tcells or enhanced CD8⁺ T-cell activity as well as blockade of mast cellupregulation of Fc receptor or prevention of degranulation. Thus, an“effective amount” will ameliorate or prevent a symptom or sign of themedical condition. Effective amount also means an amount sufficient toallow or facilitate diagnosis.

The term “treat” or “treatment” refers to a method of reducing theeffects of a disease or condition. Treatment can also refer to a methodof reducing the underlying cause of the disease or condition itselfrather than just the symptoms. The treatment can be any reduction fromnative levels and can be, but is not limited to, the complete ablationof the disease, condition, or the symptoms of the disease or condition.

A “chemotherapeutic” agent is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustardssuch as chiorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; Xeloda® Roche, Switzerland; ibandronate; CPT11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoic acid; esperamicins; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston); and antiandrogens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. The term “conjugate,” “conjugated,” “conjugation,” or“conjugating” as used in this application refers to linking of two ormore parts of a molecule together. The linking occurs by means of acovalent bond (such as peptide bond).

IL-10 Variant Proteins

The IL-10 variant molecule of the present application includesmodifications to any form of IL-10. These modifications to the IL-10molecule include additions, deletions, and/or substitution of one ormore amino acids in regions and/or domains implicated in IL-10 receptorbinding and/or those involved in imparting an inter-domain orinter-homodimeric angle in the IL-10 molecule. Exemplary IL-10 sequencesthat may be of use in constructing the variant IL-10 molecules of thepresent application include, but are not limited to, homologues fromEpstein-Barr virus (“EBV”; see, e.g., Moore et al., Science (1990)248:1230-1234; Hsu et al., Science (1990) 250:830-832; Suzuki et al., J.Exp. Med. (1995) 182:477-486), cytomegalovirus (“CMV”; see, e.g.,Lockridge et al., Virol. (2000) 268:272-280; Kotenko et al., Proc. Natl.Acad. Sci. USA (2000) 97:1695-1700), and equine herpesvirus (see, e.g.,Rode et al., Virus Genes (1993) 7:111-116), the OrF virus (see, e.g.,Imlach et al., J. Gen. Virol. (2002) 83:1049-1058 and Fleming et al.,Virus Genes (2000) 21:85-95). Other representative IL-10 sequencesinclude sequences described in NCBI accession numbers NM010548,AF307012, M37897, M84340 (mouse sequences); U38200 (equine); U39569,AF060520 (feline sequences); U00799 (bovine); U11421, Z29362 (ovinesequences); L26031, L26029 (macaque sequences); AF294758 (monkey);U33843 (canine); AF088887, AF068058 (rabbit sequences); AF012909,AF120030 (woodchuck sequences); AF026277 (possum); AF097510 (guineapig); U11767 (deer); L37781 (gerbil); AB107649 (llama and camel).

In one embodiment, the IL-10 variant molecules described herein areobtained by modifying the wild-type protein of human (SEQ ID NO.:1), CMV(SEQ ID NO.: 5), EBV (SEQ ID NO.:3) IL-10 sequences, or mouse (SEQ IDNo: 7). Representative examples of various IL-10 variant molecules areprovided in SEQ ID Nos. 9-23.

Modifications, relative to wild-type IL-10, comprise additions,deletions, and/or substitutions of one or more amino acids in theregions responsible for (i) IL-10 receptor binding and/or (ii) theformation of the inter-domain or inter-homodimeric angle of the IL-10molecule.

Variant IL-10 Molecules: IL-10 Receptor Binding Regions

The regions responsible for receptor binding include any amino acidportion located within regions that are directly involved or responsiblefor the binding of IL-10 to the IL-10 receptor 1 (IL10R1) and/or IL-10receptor 2 (IL10R2). These regions may include, for example,discontinuous portions of the IL-10 molecule previously discussed andmapped in the art (see, e.g., Yoon 2005; Josephson 2001). For example,modifications to any region, such as, but not limited to, helix A, helixF, and AB loop, responsible for forming the contact points and cleftsassociated with IL-10 binding to the IL-10 receptor are envisioned inthis application. In a preferred embodiment, the modification (e.g.,addition, deletion, and/or substitution) to the receptor binding domainincludes amino acid 31 and/or 75 of SEQ ID No. 3. In a particularlypreferred embodiment, the modifications include substituting the valineat position 31 to a leucine (V31L , herein termed “DV05”) in SEQ ID No.3, substituting the alanine at position 75 to an isoleucine (A75I,herein termed “DV06”) in SEQ ID No. 3, or both the V31L and A75Isubstitutions (herein termed “DV07”) in SEQ ID No. 3. In one aspect,DV05 is SEQ ID No: 55, DV06 is SEQ ID No: 57, and DV07 is SEQ ID No: 59.

In one embodiment, the modifications to the receptor binding domaininclude any one or more of the site Ia and/or site lb interface contactpoints discussed by Josephson et al (Immunity, 2001, 15, p. 35-46, FIG.1). In one embodiment of the application, the site Ia interface contactpoints include one or more amino acids located in the bend of helix Fand in the AB loop. In another embodiment, the site lb interface contactpoints include one or more amino acids located in the N-terminus ofhelix A and the C-terminus of helix F. In another embodiment, any one ormore amino acids located on IL-10 responsible for receptor binding willinclude any one or more of 1-10 amino acids within helix A and 1-7 aminoacids within AB loop. In another embodiment, the receptor binding regionmay include one or more modifications of the following amino acids or1-10 amino acids centered around thereof, wherein the amino acids areGlu-142, Lys-138, Asp-144, Gln-38, Ser-141, Asp-44, Gln-42, Gln-38,Arg-27, Glu-151, Arg-24, Pro-20, Ile-158, or any combination thereof.

In one aspect, the modifications to the receptor binding domain includeany one or more of the site IIa and/or site IIb interface contact pointsdiscussed by Josephson et al (Immunity, 2001, 15, p. 35-46, FIG. 1). Inone embodiment of the application, the site IIa interface contact pointsinclude one or more amino acids located in the DE loop. In anotherembodiment, the receptor binding region may include one or moremodifications of the following amino acids or 1-10 amino acids centeredaround thereof, wherein the amino acids are Ser-11, Thr-13, Asn-18,Arg-104, Arg-107, or any combination thereof. In one embodiment, thevariant IL-10 molecule will comprise 1-100 (or any integer therein)amino acid additions, deletions, and/or substitutions that impact thereceptor binding domain, wherein such additions, deletions, and/orsubstitutions either increase or decrease binding affinity of thevariant IL-10 molecule to the IL-10 receptor.

Variant IL-10 Molecules: Inter-Domain Angle Modifications

The regions responsible for the formation of the inter-domain (orinter-homodimeric, which is used interchangeably) angle of the IL-10molecule include any amino acid portion located within regions that aredirectly involved or responsible for the formation of specificinterdomain angles of IL-10 homodimers. Wild-type IL-10 forms an“L-shaped” dimer when two monomeric units of IL-10 intertwine inanti-parallel fashion. The resulting interdomain angle for human IL-10and EBV-IL10 is reported to be approximately 89 and 97 degrees,respectively. In order to tune the signal transduction of the IL-10receptor, in one embodiment, the present application seeks to modify theamino acids within the DE loop, portions of helix D or helix Eresponsible for the formation of the L-shaped dimer in each monomerresponsible for the formation of the inter-domain angle. In anotherembodiment, the regions responsible for the interdomain angle includesthe about 12 amino acid linker region located between helix D and helixE of the IL-10 protein. Modifications, by addition, deletion, orsubstitution, result in a constrained or relaxed IL-10 inter-domainangle when compared to either human IL-10 or EBV-IL10. When themonomeric IL-10 molecule is modified, resulting in aconstrained/tight/closed or relaxed/loose/opened IL-10 inter-domainangle when homodimerized, the modified IL-10 molecule will produce avariant IL-10 molecule that has an altered inter-domain angle thatengages and modulates its cognate receptor (IL-10 receptor). In anotherembodiment, the substitution includes introducing a proline within theamino acid segment located between the D helix and E helix of EBV-IL10and/or between the C helix and the D helix.

Thus, in an embodiment, the variant IL-10 molecule will comprise one ormore additions, deletions, and/or substitutions that exhibit an alteredinter-molecular angle or altered inter-domain angle, when compared tothe wild-type IL-10. The altered inter-molecular angle or alteredinter-domain angle can dimerize with an identical or different variantIL-10 molecule to result in a variant IL-10 molecule that engages theIL-10 receptor with a different angle of engagement when compared to thewild-type IL-10 molecule. The variant IL-10 molecule's different angleof engagement results in an ability to modulate or “tune” the signaltransduction of the IL-10 receptor to either activate or suppressinflammation and/or immune responses. In a preferred embodiment, thevariant IL-10 molecule is an EBV-IL10. In another preferred embodiment,the variant IL-10 molecule uses the EBV-IL10 molecule as a basis formodification. In yet another embodiment, the variant IL-10 molecule is ahybrid molecule taking portions and domains from other IL-10 molecules(such as but not limited to human IL-10, mouse IL-10, and/or CMV-IL10).

In another preferred embodiment, the variant IL-10 molecule produces arelaxed inter-domain angle that will suppress inflammatory cell (myeloidlineage cells) response, and not drive the activation of lymphocyticcells, such as T-cells. When coupled with modifications to the receptorbinding domain, preferably modifications that result in lowered orunchanged receptor affinity, the variant IL-10 molecules with therelaxed inter-domain angle will be effective in suppressing myeloidcells (monocytes, macrophages, neutrophil, granulocyte, mast cells,Kupffer cells) cytokine secretion in response to pro-inflammatorystimuli. This configuration of the variant IL-10 molecule is useful, forexample in treating inflammatory diseases such as, but not limited toIBD, Crohn's disease, psoriasis, rheumatoid arthritis, NAFLD, and NASH.

In another preferred embodiment, the variant IL-10 molecule produces aconstrained inter-domain angle that will enhance the activation ofimmune cells, such as T-cells. When coupled with modifications to thereceptor binding domain, preferably modifications that result in higherreceptor affinity, the variant IL-10 molecules with the constrainedinter-domain angle will be effective in enhancing, for example CD8⁺T-cells, NK cells, and Kupffer cell scavenging. This configuration ofthe variant IL-10 molecule is useful, for example, in treating a varietyof solid and hematological cancers including metastatic cancers.

The regions responsible for the formation of the inter-domain angle maybe continuous or discontinuous portions located within the IL-10molecule. In one embodiment, the inter-domain angle for the IL-10variant molecule will have a degree change of 1-25 degrees, in anotherpreferred embodiment, the degree change is 1-10 degrees, in a morepreferred embodiment, the degree change is 1-5 degrees, in a mostpreferred embodiment, the degree change is less than 5 degrees. In oneembodiment, the variant IL-10 molecule will comprise 1-100 (or anyinteger therein) amino acid additions, deletions, and/or substitutionsthat impact the inter-domain angle.

In one embodiment of the application, the variant IL-10 molecules willbe designed and created with the assistance of computer-based modelingto predict the region or regions most responsible for IL-10 receptorbinding and/or the inter-domain angles. The computer-based modeling willassist in providing a faster and more efficient means of predicting theregions that will benefit the most from the modifications to thereceptor binding domain and/or the inter-domain angles.

In another embodiment, the molecule of the present application includederivatives of the variant IL-10 molecules. These might includemodifications to the variant molecule to include entities that increasethe size, half-life, and bioavailability of the variant molecules.

Variant IL-10 Molecules: PEG Modifications

In one embodiment, the variant IL-10 molecules may include the additionof polyethylene glycol (PEG). PEGylated IL-10 variants will include theattachment of at least one PEG molecule. Without being bound to anyparticular theory, attachment of PEG to the IL-10 variant might protectagainst proteolysis, decrease immunogenicity, facilitate destabilizationof the IL-10 variant on the receptor to maintain its suppressive effectson myeloid cells and prevent activation of T cells.

In its most common form, PEG is a linear or branched polyetherterminated with hydroxyl groups and having the general structure:

HO—(CH₂CH₂O)_(n)—CH₂CH₂—OH

Methods of coupling PEG to variant IL-10 molecules of the presentapplication will follow those techniques/protocols already establishedin the art. For example, conjugating or coupling PEG requires activatingthe PEG by preparing a derivative of the PEG having a functional groupat one or both termini. The most common route for PEG conjugation ofproteins has been to activate the PEG with functional groups suitablefor reaction with lysine and N-terminal amino acid groups. Inparticular, the most common reactive groups involved in coupling of PEGto polypeptides are the alpha or epsilon amino groups of lysine.

The reaction of a PEGylation linker with a variant IL-10 molecule leadsto the attachment of the PEG moiety predominantly at the followingsites: the alpha amino group at the N-terminus of the protein, theepsilon amino group on the side chain of lysine residues, and theimidazole group on the side chain of histidine residues. In someembodiments, because the variant IL-10 molecules are recombinantproteins that possess a single alpha and a number of epsilon amino andimidazole groups, numerous positional isomers can be generated dependingon the linker chemistry.

Two widely used first generation activated monomethoxy PEGs (mPEGs) weresuccinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992)Biotechnol. Appl. Biochem 15:100-114; and Miron and Wilcheck (1993)Bioconjug. Chem. 4:568-569) and benzotriazole carbonate PEG (BTC-PEG;see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), which reactpreferentially with lysine residues to form a carbamate linkage, but arealso known to react with histidine and tyrosine residues. The linkage tohistidine residues on IFNα has been shown to be a hydrolyticallyunstable imidazolecarbamate linkage (see, e.g., Lee and McNemar, U.S.Pat. No. 5,985,263, which are incorporated by reference in theirentirety).

Second generation PEGylation technology has been designed to avoid theseunstable linkages as well as the lack of selectivity in residuereactivity. Use of a PEG-aldehyde linker targets a single site on theN-terminus of a polypeptide and/or protein subunit through reductiveamination. IL-10 may be PEGylated using different types of linkers andpH to arrive at a various forms of a PEGylated molecule (see, e.g., U.S.Pat. Nos. 5,252,714, 5,643,575, 5,919,455, 5,932,462, 5,985,263,7,052,686, which are all incorporate by reference in their entirety).

IL-10 Mimetic Molecules

In another embodiment, the present application includes mimeticmolecules that mirror the biological function of the IL-10 variantmolecules. These mimetics include, but are not limited to, peptides,small molecules, modified hormones, and antibodies that have structuresand or functions that are same or substantially the same as thoseproduced from the variant IL-10 molecules. IL-10 mimetic molecules thatmay form the basis for modification to replicate or mirror the IL-10variant molecules include those described in US20080139478,US20120238505, and/or US20150218222, all of which are incorporated byreference in their entirety.

IL-10 Hybrid Molecules and IL-10 Fusion Proteins

In another embodiment, the present application includes IL-10 variantmolecules that are hybrid molecules composed of portions obtained fromhuman IL-10, EBV-IL10, and/or CMV-IL10. For example, different domainswithin each of human IL-10, EBV-IL10 and/or CMV-IL10 may be combinedtogether to create a hybrid molecule, such that the combination adoptsall or portions of the receptor binding domain and/or the domainsresponsible for the interdomain angle in IL-10.

In one other embodiment, the variant IL-10 molecule is part of anengineered fusion protein. The linker or spacer can be a random aminoacid sequence (such as SSGGGGS (SEQ ID No.: 30, GGGGSGGGGSGGGGS (SEQ IDNo.: 31) or SSGGGGSGGGGSGGGGS (SEQ ID No. 54)), constant region of anantibody, a scFv or a diabody. The constant region can be derived from,but not limited to IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE. Thelinker or spacer can be preferably the constant heavy (CH) region 1,CH2, or CH3. In a more preferred embodiment, the linker of spacer is arandom amino acid sequence of SEQ ID Nos: 30 and/or 31. In anotheraspect, the linker or spacer may further comprise at least twointerchain disulfide bonds.

The fusion protein may also include at least one monomer of IL-10 orIL-10 variant molecule conjugated at the fusion protein's N-terminalend, the C-terminal end, or both. In another embodiment, the fusionprotein comprising IL-10 or IL-10 variant may also include at least onecytokine conjugated at the terminal end opposite of the IL-10 or variantIL-10 and includes IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10,IL-10 variant molecule, IFN-alpha, TGF-beta, basic-FGF, EGF, PDGF, IL-4,IL-11, or IL-13 or any combination thereof. In some preferredembodiments, the fusion protein comprises two monomeric forms of IL-10or IL-10 variant molecules conjugated at the N-terminal end of thefusion protein and two IL-10 or IL-10 variant molecules conjugated atthe C-terminal end of the fusion protein; the fusion protein comprisestwo monomeric forms of IL-10 or IL-10 variant molecules conjugated atthe N-terminal end of the fusion protein and at least one IL-2 moleculesconjugated at the C-terminal end of the fusion protein; the fusionprotein comprises two IL-10 or IL-10 variant molecules conjugated at theN-terminal end of the fusion protein and at least one IL-15 moleculesconjugated at the C-terminal end of the fusion protein. In anotherembodiment, the C-terminal end of the fusion protein may have at leasttwo different cytokines selected from IL-2, IL-7, IL-15, IL-26, IL-27,IL-28, IL-29, IL-10, IL-10 variant molecule, IFN-alpha, TGF-beta,basic-FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

In another embodiment, the fusion protein is fabricated using a singlechain variable fragment (scFv), a diabody, Fab, or any antibody fragmentas the base scaffold onto which one monomer or two monomers of IL-10,one monomer or two monomers of a IL-10 variant molecule, IL-2, IL-7,IL-15, IL-26, IL-27, IL-28, IL-29, IFN-alpha, TGF-beta, basic-FGF, EGF,PDGF, IL-4, IL-11, or IL-13, or combinations thereof are conjugated.

In one particularly preferred embodiment, the fusion protein comprisesat least one variable region, having a variable heavy chain (VH) and/orvariable light chain (VL), linked to an IL-10 or IL-10 variant molecule.In this configuration, the fusion protein comprises an IL-10 monomer orvariant IL-10 monomer linked to at least one variable region of anantibody. In one aspect, this fusion protein is a linear contiguoussequence comprising an IL-10 monomer or IL-10 monomer variant moleculelinked to a VH, linked to a VL, linked to an IL-10 monomer The variableregion of the antibody can be a variable heavy (VH) chain region, avariable light (VL) chain region, or both. A first fusion proteincomprises a protein sequence having a linear contiguous configurationsuch that an IL-10 monomer or a variant IL-10 monomer is conjugated to avariable region's (VH or VL or both) carboxy terminal end. A secondfusion protein may comprise a protein sequence having a linearcontiguous configuration such that an IL-10 monomer or a variant IL-10monomer is linked to a variable region's (VH or VL or both) aminoterminal end. A representative example of the first fusion proteindescribed above may include the following configuration:

_(NH2)(Ab1VL)_(COOH)-(linker)-_(NH2)(monoIL10)_(COOH)   a)

A representative example of the second fusion protein described abovemay include the following configuration:

_(NH2)(monoIL10)_(COOH)-(linker)_(NH2)(Ab₁VH)_(COOH)   b)

Together, the first (a) and second (b) fusion proteins form a functionalprotein complex in an anti-parallel manner, whereby the terminallylinked monomers of IL-10 or variant IL-10 form a functional homodimerand the variable regions together are capable of forming a functionalantigen binding site (“ABS”) (see e.g., FIGS. 9(a)-(f)).

In an alternative embodiment, the IL-10 monomer or variant IL-10 monomermay be conjugated to at least two variable regions from the sameantibody or from two different antibodies. In this configuration, the atleast two variable regions are a VH and VL. An example of such aconfiguration would include a first fusion protein with a linearcontiguous protein sequence of a VH region of a first antibody linked atits carboxy terminal end to an amino terminal end of a VL region of thesecond antibody subsequently linked to the amino terminal end of amonomer of IL-10 or a monomer of an IL-10 variant molecule. Analternative configuration would include a second fusion protein with alinear contiguous protein sequence of a monomer IL-10 or a monomer of anIL-10 variant molecule linked at its carboxy terminal end to an aminoterminal end of a VH region of the second antibody subsequently linkedto an amino terminal end of a VL region of the first antibody. Arepresentative example of the first fusion protein described above mayinclude the following configuration:

_(NH2)(Ab₁-VH)_(COOH)-(linker)-_(NH2)(Ab₂VL)_(COOH)-(linker)-_(NH2)(monoIL10)_(COOH)  a)

A representative example of the second fusion protein described abovemay include the following configuration:

_(NH2)(monoIL10)_(COOH)-(linker)-_(NH2)(Ab₂VH)_(COOH)-(linker)-_(NH2)(Ab₁-VL)_(COOH)  b)

Together, the first (a) and second (b) fusion proteins form a functionalprotein complex in an anti-parallel manner, whereby the terminallylinked monomers of IL-10 or variant IL-10 form a functional homodimerand the variable regions together are capable of forming an ABS (seee.g., FIGS. 9(a)-(c)).

In yet another embodiment, the fusion protein comprises two monomers ofIL-10 or two monomers of variant IL-10 that are fused together and oneor more VH and VL regions. Each monomer is individually linked to one ormore VH region and/or VL region of an antibody. When more than one VHand/or VL region is used in this fusion protein configuration, the VHand VL regions may be from the same antibody or from at least twodifferent antibodies. In one particular configuration of this fusionprotein, the VH or VL region is linked to the amino terminal end of afirst monomer which is then linked by its carboxy end to the aminoterminal end of a second monomer which is then linked to the aminoterminal end of a VL or VH. Optionally, additional VH or VL regions maybe linked to the amino or carboxy terminal ends, wherein the VH or VLregions may be from the same antibody or from a different antibody.Representative examples of the fusion protein described above mayinclude the following configurations (see e.g., FIGS. 10(d)-(f)):

_(NH2)(Ab₁-VH)_(COOH-)-(linker)-_(NH2)(monoIL10)_(COOH)-(linker)-_(NH2)(monoIL10)_(COOH-)(linker)_(-NH2)(Ab₁-VL)_(COOH)

The fusion protein described above will be capable of folding in amanner that allows the monomers of IL-10 to form a homodimer and thevariable domains (VH and VL) of an antibody to form a functional ABS.

In another embodiment, the fusion protein comprises two monomers ofIL-10 or two monomers of variant IL-1β located at the opposing terminalends of the fusion protein and at least one VH and VL region, whereinthe VH and VL regions are linked together. In this configuration, the VHand VL regions are fused together and each monomer is individuallylinked to either a VL region or a VH region of a first antibody. In thisconfiguration, the IL-10 monomers or the monomers of variant IL-10 areeach individually linked to either a VH or VL of a first antibody. Arepresentative example of the fusion protein described above may includethe following configurations (see, e.g., FIGS. 10(a)-(c)):

_(NH2)(monoIL10)_(COOH)-(linker)_(-NH2)(Ab₁-VH)_(COOH-)(linker)-_(NH2)(Ab₁-VL)_(COOH-)(linker)_(-NH2)(monoIL10)_(COOH)  a)

_(NH2)(monoIL10)_(COOH)-(linker)_(-NH2)(Ab₁-VL)_(COOH-)(linker)-_(NH2)(Ab₁-VH)_(COOH-)(linker)_(-NH2)(monoIL10)_(COOH)  b)

The monomer of IL-10 or the monomer of a variant IL-10 may be linked tothe VH or VL sequence through a linker sequence. The linker can be acarboxy terminal linker linking a carboxy end of a variable chain region(VH or VL) to an amino terminal end of a monomer of IL-10 or a monomericIL-10 variant molecule. Alternatively, the linker can be an aminoterminal linker whereby the carboxy terminal end of a monomeric IL-10 ora monomeric IL-10 variant molecule is linked to the amino terminal endof a variable chain region (VH or VL).

Thus, in one form, the fusion protein comprises a monomeric IL-10molecule or a variant IL-10 molecule linked to two variable regions fromat least two different antibodies, wherein the two variable regions areconfigured as a VH region from a first antibody linked to a VL regionfrom a second antibody or a VL from the first antibody linked to a VHfrom the second antibody. The fusion protein according to this form mayinclude monomeric IL-10 molecule or a variant thereof including at leastone amino acid substitution that increases or decreases affinity to anIL-10 receptor. The amino acid substitution impacting IL-10 receptorbinding may occur in a human, CMV or EBV IL-10. The amino acidsubstitution may preferably be in an EBV IL-10 and include amino acidsubstitution at position 31, 75, or both. The amino acid substitutionsmay include any one or more of a V31L or A75I substitution or both. Inaddition to the amino acid substitutions impacting IL-10 receptorbinding affinity, the IL-10 variant may also include modifications thatimpact the inter-domain angle. In another embodiment, the fusion proteincomprises a configuration selected from: (a) a VH region of the firstantibody linked at its carboxy terminal end to an amino terminal end ofa VL region of the second antibody subsequently linked to a carboxyterminal end of a monomer of IL-10 or a variant thereof; or (b) an IL-10molecule or a variant thereof linked at its carboxy terminal end to anamino terminal end of a VH region of the second antibody subsequentlylinked to an amino terminal end of a VL region of the first antibody.These fusion protein configurations include, in one preferred embodimenta sequence of SEQ ID Nos.: 24-28, 29, and 33-53. These fusion proteinssequences are capable of forming a complex wherein the monomers of IL-10or monomers of variant IL-10 are capable of forming a homodimer. Such acomplex may include and/or configured as a diabody complex.

In another form, the fusion protein may be fashioned as animmunoconjugate comprising a first fusion protein comprising at itsamino terminal end a heavy chain variable region (VH) of a firstantibody linked to a light chain variable region (VL) of a secondantibody further linked to a monomer of IL-10; and a second fusionprotein comprising at its amino-terminal end a monomer of IL-10 linkedto a VH of the second antibody further linked to a VL of the firstantibody, wherein the VH and VL of the first and second antibodiesassociate into a diabody and the monomers of IL-10 form a functionaldimeric IL-10 molecule. In another preferred embodiment, animmunoconjugate complex comprises a first fusion protein comprising atits amino terminal end a VH region of a first antibody and a monomericIL-10 molecule linked by its amino terminal end; and a second fusionprotein comprising at its amino terminal end a monomer of IL-10 linkedto a VL of the first antibody, wherein the VH region of the firstantibody associates with the VL region of first antibody therebyallowing the monomeric IL-10 molecules on each peptide chain to form afunctional IL-10 dimer. The monomer(s) of IL-10 or monomer(s) variantIL-10 may include, as described above, amino acid modifications thatimpact IL-10 receptor binding and/or inter-domain angle. In anotherpreferred embodiment, an immunoconjugate complex comprises a firstfusion protein comprising at its amino terminal end a VH region of afirst antibody linked to a monomeric IL-10 molecule; and a second fusionprotein comprising at its amino terminal end a monomer of IL-10 linkedto a VL of the first antibody, wherein the VH region of the firstantibody associates with the VL region of first antibody therebyallowing the monomeric IL-10 molecules on each peptide chain to form afunctional IL-10 dimer. In yet another embodiment, the immunoconjugatecomprises at its amino terminal end a monomer of a first IL-10 (or IL-10variant molecule) monomer linked to a VH region of a first antibodylinked to a VL region of the first antibody linked to a monomer of asecond IL-10 (or IL-10 variant molecule), wherein the two IL-10 monomersare able to associate together to form a functional dimer of IL-10. TheVH and VL regions described are capable of forming an antigen bindingsite that specifically target an antigen (e.g., receptor, protein,nucleic acid, etc.). Thus, there are two chains, Chain 1 and Chain 2which together for a fusion protein complex that creates a functioningIL-10 (or IL-10 variant molecule) homodimer. Representative fusionprotein chains (i.e., Chain 1 and Chain 2) include the following:

Chain 1 Chain 2 SEQ ID No: 24 SEQ ID No: 25 SEQ ID No: 26 SEQ ID No: 27SEQ ID No: 28 SEQ ID No: 29 SEQ ID No: 35 SEQ ID No: 36 SEQ ID No: 38SEQ ID No: 39 SEQ ID No: 41 SEQ ID No: 42 SEQ ID No: 46 SEQ ID No: 47SEQ ID No: 48 SEQ ID No: 49 SEQ ID No: 50 SEQ ID No: 51

The fusion proteins include a VH and VL pair from at least one antibody.The VH and VL pair act as a scaffolding onto which monomers of IL-10 orvariants thereof may be attached such that may be able to homodimerizeinto a functioning IL-10 molecule. A person of skill in the art willtherefore appreciate that the VH and VL scaffolding used in the fusionprotein can be selected based on the desired physical attributes neededfor proper IL-10 or IL-10 variant protein dimerization and/or the desireto maintain VH and VL targeting ability. Likewise, a person of skillwill also understand that the CDR regions within the VH and VL pair mayalso be substituted with other CDR regions to obtain a specificallytargeted fusion protein. It is also envisioned that if the fusionprotein is not intended to target any specific antigen, a VH and VL pairmay be selected as the scaffolding that does not target any particularantigen (or is an antigen in low abundance in vivo), such as the VH andVL pair from an anti-HIV and/or anti-Ebola antibody. The fusion proteinmay comprises a range of 1-4 variable regions. The variable regions maybe from the same antibody or from at least two different antibodies. Theantibody variable chains can be obtained or derived from a plurality ofantibodies (e.g., those targeting proteins, cellular receptors, and/ortumor associated antigens, etc.). In another embodiment, the variableregions are obtained from antibodies that target antigens associatedwith various diseases (e.g., cancer) or those that are not typicallyfound or rarely found in the serum of a healthy subject, for examplevariable regions from antibodies directed to EGFR, PDGFR, VEGFR,Her2Neu, FGFR, GPC3, or other tumor associated antigens, MadCam, ICAM,VCAM, or other inflammation associated cell surface proteins, HIV and/orEbola. Thus, in one embodiment, the variable regions are obtained orderived from anti-EGFR, anti-MadCam, anti-HIV (Chan et al, J. Virol,2018, 92(18):e006411-19), anti-ICAM, anti-VCAM, or anti-Ebola (USPublished Application 2018/0180614, incorporated by reference in itsentirety, especially mAbs described in Tables 2, 3, and 4) antibodies,for example. In another embodiment, the variable regions are obtained orderived from antibodies capable of enriching the concentration ofcytokines, such as IL-10, to a specific target area so as to enableIL-10 to elicit its biological effect. Such an antibody might includethose that target overexpressed or upregulated receptors or antigens incertain diseased regions or those that are specifically expressed incertain impacted areas. For example, the variable regions might beobtained from antibodies specific for epidermal growth factor receptor(EGFR); CD52; various immune check point targets, such as but notlimited to PD-L1, PD-1, TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2;HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2;EDB-FN; TGFβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4integrin SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1;SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1 to name a few. Amonomer of IL-10 (e.g., human, CMV, or EBV) or variant IL-10 molecule(described herein) is conjugated to either the amino terminal end or thecarboxy terminal end of a variable region (VH or VL), such that theIL-10 or variant IL-10 molecule is able to dimerize with one another.

The fusion protein or fusion protein complex may also have an antigentargeting functionality. The fusion protein or fusion protein complexwill comprise VH and VL regions that are able to associate together toform an antigen binding site or ABS. In some configurations, the IL-10or IL-10 variant molecule or monomers thereof will be covalently linkedto the end comprising the antigen binding site. These targeting fusionproteins may comprise at least one functioning variable region or pairedVH and VL at one end of the fusion protein such that the fusion proteinretains the capacity to target an antigen as well as having afunctioning homodimer of an IL-10 or IL-10 variant molecule (see, FIGS.9(a)-(f) and 10(a)-(f)). The variable regions may be further modified(e.g., by addition, subtraction, or substitution) by altering one ormore amino acid that reduce antigenicity in a subject. The VH and VLpair form a scaffolding onto which CDR regions obtained for a pluralityof antibodies can be grafted. Such antibody CDR regions include thoseantibodies known and described above. For example, the CDR regions fromany antibody may be grafted onto a VH and VL pair such as thosedescribed in SEQ ID Nos: 37, 44, or 45 or those fusion proteins that arecapable of forming a fusion protein complex such as those described inSEQ ID Nos: 46 and 47; 48 and 49; or 50 and 51. The CDR regions in theabove described VH and VL scaffolding will include the following numberof amino acid positions available for CDR engraftment/insertion:

Heavy chain CDR1 3-7 amino acids Heavy chain CDR2 7-11 amino acids Heavychain CDR3 7-11 amino acids Light chain CDR1 9-14 amino acids Lightchain CDR2 5-9 amino acids Light chain CDR3 7-11 amino acids

In another aspect, the fusion protein described above may be representedby one of the following general formula:

1) IL10-L¹-X¹-L¹-X²-L¹-IL10   (Formula I);

2) (Z)_(n)—X¹-L²-Y²-L¹-IL10   (Formula II);

3) IL10-L¹-Y¹-L²-X²—(Z)_(n)   (Formula III);

4) X¹-L²-X²-L¹-IL10   (Formula IV);

5) IL10-L¹-X¹-L²-X²   (Formula V);

6) X¹-L¹-IL10   (Formula VI); and

7) IL10-L¹-X²   (Formula VII)

wherein

-   -   “IL-10” is human Il-10 (SEQ ID No: 1); EBV IL-10 (SEQ ID No: 3),        DV05 (SEQ ID No:14, 18, or 55), DV06 (SEQ ID No: 15, 19, or 57),        or DV07 (SEQ ID No:16, 20, or 59), in a preferred embodiment,        “IL-10” consists of DV05, DV06, or DV07, more preferably “IL-10”        consists of SEQ ID Nos: 55, 57, or 59;    -   “L¹” is a linker of SEQ ID No: 31 or 54;    -   “L²” is a linker of SEQ ID No: 30;    -   “X¹” is a VH region obtained from a first antibody specific for        epidermal growth factor receptor (EGFR); CD52; various immune        check point targets, such as but not limited to PD-L1, PD-1,        TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2; HER2; EpCAM; ICAM        (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TFGβ        Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin        SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1;        SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola;    -   “X²” is a VL region obtained from the same antibody as X₁;    -   “Y¹” is VH region obtained from a second antibody specific for        epidermal growth factor receptor (EGFR); CD52; various immune        check point targets, such as but not limited to PD-L1, PD-1,        TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2; HER2; EpCAM; ICAM        (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TFGβ        Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin        SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C 1; SR-D1; SR-E1;        SR-F 1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola;    -   “Y²” is a VL region obtained from the same antibody as Y₁;    -   wherein X and Y are obtained from the same or different        antibody;    -   “Z” is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3,        IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29,        GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis        factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13;    -   “n” is an integer selected from 0-2.

In an embodiment, the substituents of Formula I-VII, above, ispreferably selected from the following: IL-10 is preferably DV05, DV06,or DV07, more preferably IL-10 consists of a DV05, DV06, or DV07 or mostpreferably IL-10 consists of SEQ ID No: 55, 57, or 59; X₁ and X2 arepreferably an anti-EGFR, anti-PDGFR, anti-FGFR, anti-VEGF, anti-Her2Neu,anti-GPC3, anti-MAdCAM, anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV, oranti-Ebola; Y1 and Y2 are preferably anti-EGFR, anti-MAdCAM,anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV, or anti-Ebola; Z isselected from IL-2, IL-7, or IL-15; and n is 1. In a most preferredembodiment, the fusion proteins are anyone of SEQ ID Nos: 33-53, 61, 63,65, or 67. Those of skill in the art will understand that the presenceof a Histidine tag is used in the purification process of the fusionprotein and maybe left intact or removed from the final product. Thoseof skill in the art will also understand that the VH and VL frameworkregions of any of the antibodies described above may be substituted withother complementary-determining regions (CDR) regions. For example, ifthe VH and VL regions are from an anti-Ebola antibody, the six CDRregions (i.e., CDRs 1-3 of both the VH and VL) may be substituted withthe 6 CDR regions of an anti-EGFR antibody (e.g., cetuximab). Thus, inone preferred embodiment, the fusion protein is SEQ ID Nos: 33-34,52, or53. In another preferred embodiment, the fusion protein is one havingthe scaffolding represented by SEQ ID Nos: 37; 44; 45; 46-47; 48-49; or50-51, where any 6 CDR regions from any antibody may be grafted. Inother preferred embodiments, the CDR regions from the VH and VL regionsof an anti-Ebola antibody may be grafted with the CDR regions from ananti-MAdCAM, anti-VCAM, or anti-ICAM-1, -2, -3, -4 antibody, wherein ina preferred embodiment the CDR regions may be grafted into a fusionprotein of SEQ ID No: 37. The fusion proteins as described in theformulas II and III; formulas IV and V; and formulas VI and VII, aboveare designed to associate together to form a biologically activehomodimer of IL-10 (or variant thereof). The fusion proteins describedabove are designed to either be non-targeting or targeting depending onthe pair of VH and VL regions selected and/or the CDR regions engraftedinto the VH and VL. The term “non-targeting” is meant to describe a VHand VL region that is not able to target to a specific antigen locatedin vivo because the antigen is not present or the antigen binding site(ABS) has been disabled or modified to eliminate the ABS functionality.

The fusion proteins described above may be further conjugated toaccessory proteins/molecules. An accessory protein as used hereindescribes a protein that is conjugated to the fusion protein or fusionprotein complex such that it is linked to the side that is opposite thatof the IL-10 monomer or variant IL-10 monomer molecule. The addition ofthe accessory protein effectively create a multifunctional molecule thatincorporates both IL-10 or IL-10 variant molecule functionality and thefunctionality of the accessory protein. Attachment of the accessoryproteins (e.g., cytokines IL-2, IL-7, IL-12, IL-15, etc.) may beattached, for example, to the fusion protein comprising the VH and VLscaffolding at the N-terminal end of VH region. For example, as itapplies to fusion proteins or fusion protein complexes for treatingoncology, the accessory protein includes, but is not limited to, IL-10,IL-10 variant molecule IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8,IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α,-β, -γ, TGF-β, or tumor necrosis factors -α, -β, basic FGF, EGF, PDGF,IL-4, IL-11, or IL-13 or any combination thereof. As it applies tofusion proteins or fusion protein complexes for treating inflammatorydiseases, the accessory protein includes, but is not limited to TGFI3.As it applies to fusion proteins or fusion protein complexes fortreating autoimmune diseases (such as but not limited to fatty liverdisease), the accessory molecule includes, but is not limited toobticholic acid, aramchol, elafibranor, liraglutide, selonsertib, orsimtuzumab. The accessory proteins defined above, when described usingformulas I-V above may be attached to the substituent X1 or Y1, at theN-terminal side of the VH portion of the scaffolding.

The fusion proteins described above may also include additional aminoacid sequences that aid in the recovery or purification of the fusionproteins during the manufacturing process. These may include varioussequence modifications or affinity tags, such as but not limited toprotein A, albumin-binding protein, alkaline phosphatase, FLAG epitope,galactose-binding protein, histidine tags, and any other tags that arewell known in the art. See, e.g., Kimple et al (Curr. Protoc. ProteinSci., 2013, 73:Unit 9.9, Table 9.91, incorporated by reference in itsentirety). In one aspect, the affinity tag is an histidine tag having anamino acid sequence of HHHFIHH (SEQ ID No.: 32). The histidine tag maybe removed or left intact from the final product. In another embodiment,the affinity tag is a protein A modification that is incorporated intothe fusion protein (e.g., into the VH region of the fusion proteinsdescribed herein), such as those described in SEQ ID Nos: 34 or 44-53. Aperson of skill in the art will understand that any fusion proteinsequence described herein can be modified to incorporate a protein Amodification by inserting amino acid point substitutions within theantibody framework regions as described in the art.

In yet another embodiment, the various fusion proteins described abovemay be used in a method of treating cancer, treating or preventing IBDor Crohn's disease, autoimmune disease, NAFLD, or NASH.

IL-10 Variant Polynucleotides

Also within the scope of the present application includes polynucleotidesequences that encode for the variant IL-10 molecules and the variousfusion proteins and/or immunocytokines described above. Uponlocalization of key IL-10 receptor binding regions and/or regions forinter-domain angle in the variant IL-10 molecules of the presentapplication, the DNA modifications necessary to effect the desiredmodifications into amino acid sequences are within the skill set of aperson of skill in the art. Such modifications will employ conventionalrecombinant DNA techniques and methods. For example, the addition orsubstitution of specific amino acid sequences may be introduced into anIL-10 sequence at the nucleic acid (DNA) level using site-directedmutagenesis methods employing synthetic oligonucleotides, which methodsare also well known in the art.

In another embodiment, the polynucleotides encoding the variant IL-10sequence can be made using standard techniques of molecular biology. Forexample, polynucleotide sequences coding for the above-described variantmolecules can be obtained using recombinant methods, such as byscreening cDNA and genomic libraries from cells expressing the gene, orby deriving the gene from a vector known to include the same. The geneof interest can also be produced synthetically, rather than cloned,based on the known sequences. The molecules can be designed withappropriate codons for the particular sequence. The complete sequence isthen assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge,Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; and Jayet al., J. Biol. Chem. (1984) 259:6311.

In one embodiment, the IL-10 variant molecule or fusion proteins thereofis a nucleic acid molecule encoding any of SEQ ID No: 9-29, 33-53, 55,57, or 59. In another embodiment, the IL-10 variant molecule is DV05,DV06, or DV07 of SEQ ID No: 56, 58, or 60, respectively. The nucleicacid molecule encoding DV05, DV06, or DV07 may include insertions,deletions, or substitutions (e.g., degenerate code) that do not alterthe functionality of the IL-10 variant molecule. The nucleotidesequences encoding the IL-10 variant and fusion proteins describedherein may differ from the sequences of SEQ ID Nos: 1, 3, 5, 7, 9-29,33-53, 55, 57, 59, 61, 63, 65, or 67 due to the degeneracy of thegenetic code and may be 70-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99%, homologous to the aforementioned sequences.Nucleotide sequence encoding the IL-10 variant and fusion proteins ofSEQ ID Nos: 1, 3, 5, 7, 9-29, 33-53, 55, 57, 59, 61, 63, 65, or 67 mayalso be 70%-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99%, homologous to due to the insertions, deletions, or substitutionsof at least one nucleotide. Also envisaged in the present applicationare nucleotide sequences that have 70-99%, preferably 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99%, sequence homology to SEQ ID Nos:2, 4, 6, 8, 56, 58, 60, 62, 64, 66, and 68, due to insertion, addition,deletion, or substitution of at least one nucleotide. Moreover, thenucleotide sequences encoding the IL-10 variant and fusion proteinsdescribed herein may further comprise well known sequences that aid in,for example, the expression, production, or secretion of the proteins.Such sequences may include, for example a leader sequence, signalpeptide, and/or translation initiation sites/sequence (e.g. Kozakconsensus sequence). The nucleotide sequences described herein may alsoinclude one of more restriction enzyme sites that allow for insertioninto various expression systems/vectors.

In another embodiment, the polynucleotides are harbored in vectorscomprising the desired IL-10 sequences or artificially synthesized usingoligonucleotide synthesis techniques known in the art, such assite-directed mutagenesis and polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, supra. In another embodiment, thenucleotide sequences encoding the variant IL-10 molecules are obtainedthrough a process of annealing complementary overlapping syntheticoligonucleotides produced in an automated polynucleotide synthesizer,followed by ligation and amplification of the ligated nucleotidesequence via PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci.USA (1991) 88:4084-4088. Additionally, oligonucleotide-directedsynthesis (Jones et al., Nature (1986) 54:75-82), oligonucleotidedirected mutagenesis of preexisting nucleotide regions (Riechmann etal., Nature (1988) 332:323-327 and Verhoeyen et al., Science (1988)239:1534-1536), and enzymatic filling-in of gapped oligonucleotidesusing T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989)86:10029-10033) can be used to provide molecules for use in the subjectmethods.

A variety of suitable expression vectors may be used and are well knownto a person skilled in the art, which can be used for expression andintroduction of the variant IL-10 molecules and fusion proteins. Thesevectors include, for example, pUC-type vectors, pBR-type vectors,pBI-type vectors, pGA-type, pBinl9, pBI121, pGreen series, pCAMBRIAseries, pPZP series, pPCV001, pGA482, pCLD04541, pBIBAC series, pYLTACseries, pSB11, pSB1, pGPTV series, and viral vectors and the like can beused.

The vectors harboring the IL-10 variant molecules may also include othervector componentry required for vector functionality. For example, thevector may include signal sequences, tag sequences, proteaseidentification sequences, selection markers and other sequencesregulatory sequences, such as promoters, required for proper replicationand expression of the variant IL-10 molecules. The particular promotersutilized in the vector are not particularly limited as long as they candrive the expression of the variant IL-10 molecule in a variety of hostcell types. Likewise, the type of Tag promoters are not be limited aslong as the Tag sequence makes for simpler or easier purification ofexpressed variant IL-10 molecule easier. These might include, forexample, 6-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus, biotin, FLAG,myc, RCFP, GFP and the like can be used. Protease recognition sequencesare not particularly limited, for instance, recognition sequences suchas Factor Xa, Thrombin, HRV, 3C protease can be used. Selected markersare not particularly limited as long as these can detect transformedrice plant cells, for example, neomycin-resistant genes,kanamycin-resistant genes, hygromycin-resistant genes and the like canbe used.

The resulting DNA constructs carrying the desired IL-10 variants orfusion protein can then be used directly in gene therapy or can be usedto produce recombinant IL-10 variant or fusion proteins. In oneembodiment, the variant IL-10 molecules or fusion protein of the presentapplication can be delivered by any method know in the art, includingdirect administration of the mutant IL-10 protein and gene therapy witha vector encoding the mutant IL-10 protein. Gene therapy may beaccomplished using plasmid DNA or a viral vector, such as anadeno-associated virus vector, an adenovirus vector, a retroviralvector, etc. In some embodiments, the viral vectors of the applicationare administered as virus particles, and in others they are administeredas plasmids (e.g. as “naked” DNA).

Other methods for the delivery of the nucleotide sequences include thosewhich are already known in the art. These would include the delivery ofthe nucleotide sequences, such as but not limited to DNA, RNA, siRNA,mRNA, oligonucleotides, or variants thereof, encoding the IL-10 or IL-10variant molecules by a cell penetrating peptide, a hydrophobic moiety,an electrostatic complex, a liposome, a ligand, a liposomalnanoparticle, a lipoprotein (preferably HDL or LDL), a folate targetedliposome, an antibody (such as Folate receptor, transferrin receptor), atargeting peptide, or by an aptamer. The nucleotide sequences encodingIL-10 variant molecules may be delivered to a subject by directinjection, infusion, patches, bandages, mist or aerosol, or by thin filmdelivery. The nucleotide (or the protein) may be directed to any regionthat is desired for targeted delivery of a cytokine stimulus. Thesewould include, for example, the lung, the GI tract, the skin, liver,brain though intracranial injection, deep seated metastatic tumorlesions via ultrasound guided injections.

Testing IL-10 Variants

In one embodiment, the variant IL-10 molecules or fusion proteinsthereof will be screened for novel functions not previously generatedusing IL-10 homodimer sequences that suppress inflammatory cytokinesecretion by macrophages but do not activate T cells. In one of thepreferred embodiments, the variant IL-10 molecule or fusion proteinsthereof will be based on an EBV-IL10 backbone that possess ananti-inflammatory response but lacks the ability to stimulate T-cells.These variant EBV-IL10 molecules or fusion proteins thereof will includemodifications to the receptor binding domains and the previouslyunexplored linkage regions that alter the primary, secondary andtertiary structure to constrain or broaden the angle between the IL-10homodimers. In another embodiment, IL-10 variant molecules comprisingmodifications to the linkage region or the regions responsible forinter-domain angle formation, will possess enhanced CD8+ T-cellfunction. In another embodiment, the IL-10 variant molecules or fusionproteins thereof comprising modifications to the linkage region or theregions responsible for inter-domain angle formation, will possesssuppressed myeloid function but enhanced Kupffer cells function.

Once the variant IL-10 molecules or fusion proteins thereof areconstructed and expressed, a person of skill in the art will be capableof performing screening assays on the IL-10 variant molecules or fusionproteins thereof to determine whether the molecules possess the desiredbiological functions imparted by the modifications to the IL-10 receptorbinding regions and/or the inter-domain angle. A plurality of screeningassays are known and available to those of skill in the art to test forthe desired biological function. In one embodiment, the desiredbiological function includes, but are not limited to, reducedanti-inflammatory response, reduce T-cell stimulation, enhanced T-cellfunction, enhanced Kupffer cell functionality and reduced mast celldegranulation.

For example, it is known that IL-10 exposure primes T cells to generateand secrete more IFNγ upon T cell receptor stimulation. Simultaneously,IL-10 exposure prevents the secretion of TNFα, IL-6 and otherpro-inflammatory cytokines secreted from monocytes/macrophages inresponse to LPS. IL-10 also suppresses FoxP3⁺CD4⁺ T_(reg) proliferation.In one embodiment, those IL-10 variants molecules or fusion proteinsthereof that maximize monocyte/macrophage suppression but lack T celleffects, including both stimulatory and suppressive responses, will bepositively selected. In one embodiment, screening for IL-10 variantmolecules or fusion proteins thereof that possess increasedanti-inflammatory effects will be positively selected for the treatmentof autoimmune, anti-inflammatory disease or both. In anotherembodiments, IL-10 variant molecules or fusion proteins thereof thatenhance Kupffer cell scavenging and lack T_(reg) suppression will alsobe selected to develop for treatment of Non-alcoholic SteatoticHepatitis (NASH) and/or Non-alcoholic Fatty Liver Disease (NAFLD). Inyet another embodiments, IL-10 variants that maximize T cell biology,including both stimulatory and suppressive responses, and also possessesenhanced Kupffer cell scavenging, will be selected to develop for thetreatment of cancer.

The literature is replete with descriptions for assay the effect ofcytokines on cells of the immune system, such as T cells,monocytes/macrophages, Kupffer cells, T_(reg) cells, and mast cells, forexample. The present application will apply these assay systems fortesting the biological response employing similar assays by contactingthe variant IL-10 molecules or fusion proteins thereof of the presentapplication.

Various methods are described in the prior art for assaying theeffectiveness of eliciting a T cell response. Any one of these methodsare applicable for testing the variant IL-10 molecules described herein.For example, Chan et al. (2015) describes one such method that isapplicable to the variant IL-10 molecules. CD8⁺ T cells are isolatedfrom peripheral blood mononuclear cells (PBMCs) using anti-CD8microbeads. The isolated CD8⁺ T cells are activated using anti-CD3 andanti-CD28 antibodies. For example, the activation may occur using platescoated with at least about 5 to 20 micro grams/mL of anti-CD3 antibodyand at least about 1 to 5 micro grams/mL anti-CD28 antibody over aperiod of about 3 days. Following activation, the T cells are collected,plated, and treated with EBV-IL10 variants or fusion proteins thereoffor a period of about 3-5 days. Commercially available PEGylatedrecombinant human IL-10 or EBV-IL10 may be used as a control. Followingtreatment with the EBV-IL10 variants, T cells were treated with solubleanti-CD3. Following treatment with anti-CD3, the cell culture media iscollected and assayed by ELISA for secretion of interferon gamma (IFNγ).

Various methods are described in the prior art for assayingmonocytes/macrophages stimulation by cytokines. Any one of these methodsare applicable for testing the variant IL-10 molecules described herein.For example, Conway et al (2017) describes one such method that isapplicable to the variant IL-10 molecules. Human monocytes are isolatedfrom buffy coats of fresh donor blood using a Ficoll gradient, followedby hypertonic density centrifugation in Percoll. After 30-mincultivation in RPMI supplemented with 5% human serum and 1% L-glutamine,the monocytes became adherent and are washed with SMEM Spinner medium toremove contaminating lymphocytes. Solutions and materials were tested toensure the absence of LPS. After 4 days of cultivation, themonocytes/macrophages are contacted or incubated with 10 ng/ml LPS anddifferent concentrations of variant IL-10 molecules for a period of atleast 24 h. Culture supernatants are harvested, and TNF-a, and IL-1βconcentrations are determined by ELISA.

Various methods are described in the prior art for assaying Kupffer cellresponse to cytokines. Any one of these methods are applicable fortesting the variant IL-10 molecules described herein. For example, Chanet al (2016) describes one such method that is applicable to the variantIL-10 molecules. Kupffer cells are plated in 24-well or 96-well platesand incubated overnight in hepatocyte incubation medium (phenol-red freeRPMI, pen/strep, Cell Maintenance Supplement B (Invitrogen)). Cells werewashed and exposed for 24 hours to variant IL-10 molecules. Cells arewashed once and exposed to 15-20 μl DiI-LDL, DiI-VLDL, DiI-OxLDL orDiI-AcLDL, 2 μl DMSO, 15 μl Cytochalasin D, where uptake is measuredafter 4 hours. All cells are washed once in 1× PBS and lysed with 110 μlcell lysis buffer. 45 μl of cell lysate is transferred to clear bottomblack walled plates where fluorescence is read at 575 nm.

Various methods are described in the prior art for assaying theeffectiveness of stimulating T regulatory cell response using cytokines.Any one of these methods are applicable for testing the variant IL-10molecules described herein. For example, Chan et al (2016) describes onesuch method that is applicable to the variant IL-10 molecules. CD4⁺ Tcells are isolated with CD4⁺ microbeads and cultured for 5-6 days inAIMV media containing various concentrations of the variant IL-10molecule and 2 micrograms/mL immobilized anti-CD3 and 1 mg/mL anti-CD28.Cells are analyzed for FoxP3 expression by flow cytometric analysis todetermine if TGF-β or IL-2 is induced in the FOX P3⁺CD4⁺ T regulatorycells.

Various methods are described in the prior art for assaying theeffectiveness of proliferating mast cells in response to cytokinestimulation. The murine mast cell line MC/9 is a common cell line usefor manufacturing release testing of IL-10 molecules. Specifically,IL-10 and IL-10 variant molecules induce dose titratable proliferationof mast cells. Conversely, IL-10 inhibits Fc expression by mast cells,suggesting IL-10 exerts both stimulatory and suppressive effects onthese cells. Any one of these methods are applicable for testing thevariant IL-10 molecules described herein. For example, Thompson-Snipeset al (1991) describes one such method that is applicable to the variantIL-10 molecules. MC/9 mast cells are plated in flat-bottomed 24-wellplates containing 1 ml of RPMI 1640, 10% FCS, 50 mM 2-ME, and varyingconcentrations of variant cytokines. After culturing for 3 days, cellare counted using a cell counter to determine the impact of the variantIL-10 molecules on mast cell proliferation.

It is known that IL-10 plays a role in inhibiting mast cell expressionof the IgE receptor, FcεRI, and IgE-mediated cytokine production. Thus,methods have been described in the prior art for testing IL-10's impacton mast cells. These methods are applicable for testing the IL-10variant molecules described herein. For example, Kennedy Norton et al(2008) describes one such method. Human mast cells were isolated fromdonor skin samples and cultured in medium containing stem cell factor(SCF) in the presence or absence of IL-10. FcεRI expression wasdetermined by flow cytometry using of anti-FcεRI specific antibodiesfollowed by FITC-labeled anti-mouse F(ab′)₂.

Compositions and Formulations Comprising IL-10 Variant Molecules

The IL-10 variant molecules or fusion proteins thereof of the presentapplication may also be formulated in a pharmaceutical compositioncomprising a therapeutically effective amount of the variant IL-10molecule and a pharmaceutical carrier and/or pharmaceutically acceptableexcipients. The pharmaceutical composition may be formulated withcommonly used buffers, excipients, preservatives, stabilizers, Thepharmaceutical composition will be formulated for administration to apatient in a therapeutically effective amount sufficient to provide thedesired therapeutic result. Preferably, such amount has minimal negativeside effects. In one embodiment, the amount of variant IL-10 molecule orfusion protein thereof administered will be sufficient to treatinflammatory diseases or condition. In another embodiment, the amount ofvariant IL-10 molecule or fusion proteins thereof administered will besufficient to treat cancer. The amount administered may vary frompatient to patient and will need to be determined by considering thesubject's or patient's disease or condition, the overall health of thepatient, method of administration, the severity of side-effects, and thelike. In a preferred embodiment, the pharmaceutical composition willinclude a variant IL-10 molecule or fusion protein thereof that includesone or both of a modification to the receptor binding domain and/or theinter-domain angle of the IL-10. In another embodiment, the variantIL-10 molecule is a PEGylated form of the variant IL-10 molecule. In yeta more preferred embodiment, the pharmaceutical composition comprisesthe variant IL-10 molecule incorporated as a fusion protein orimmunocytokine and pharmaceutical excipients.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside effects. The appropriate dose administered to a patient istypically determined by a clinician using parameters or factors known orsuspected in the art to affect treatment or predicted to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.

The dose administered to the patient may also be optimized based on theaddition of certain serum half-life extension modifications to the IL-10or variant IL-10 molecule (see, e.g., pegylated IL-10, discussed indetail, for example, in U.S. Pat. Nos. 9,943,568, 10,010,588, and10,143,726, has been known to improve circulation half-life of IL-10).The fusion protein, immunoconjugates, fusion protein, minobodies, anddiabodies comprising IL-10 or variant IL-10, disclosed herein, are alsoable to extend circulation half-life, while simultaneously retaininghigh affinity binding to the IL-10 receptor. Thus, in one embodiment,various diseases, disorders, or conditions associated with IL-10 or thatmay be improved by administering a fusion protein, a complex of fusionproteins, immunoconjugate, or a diabody comprising IL-10 or a variantIL-10, may be administered to a patient in need thereof. In onepreferred embodiment, diseases, disorders, or conditions associated withIL-10 may be treated or prevented by administering to a patient in needthereof a therapeutically effective amount of an immunoconjugatecomplex, fusion protein, or diabody with an EBV IL-10 or a variantthereof (including a variant impacting IL-10 receptor biding affinity),wherein the immunoconjugate complex, fusion protein, or diabody has amolecular weight of about 60 to 155 kDa, wherein the therapeuticallyeffective amount is in the range of about 0.5 microgram/kilogram to 100micrograms/kilogram. The immunoconjugate, fusion protein, or diabodydescribed herein may be administered daily, three times a week, twice aweek, weekly, bimonthly, or monthly. The EBV IL-10 portion and thevariable regions of the immunoconjugate, fusion protein, or diabody maybe any configuration or combination of those structures discussedherein. The half-life extended molecules described herein will beeffective in treating a variety of diseases including, but not limitedto, cancer, inflammatory diseases, autoimmune diseases (e.g.,nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease(NAFLD)), and elevated cholesterol.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic,anti-inflammatory agents, or radiation, are well known in the art. Thesemight include combination treatments with other therapeutic agents, suchas but not limited to one or more the following: interferon-β, forexample, IFNβ-1α and IFN-β-1 β; a protein that simulates myelin basicprotein; corticosteroids; IL-1 inhibitors; TNF inhibitors; anti-TNFαantibodies, anti-IL-6 antibodies, IL-1br-Ig fusion, anti-IL-23antibodies, antibodies to CD40 ligand and CD80; antagonists of IL-12 andIL-23, e.g., antagonists of a p40 subunit of IL-12 and IL-23 (e.g.,inhibitory antibodies against the p40 subunit); IL-22 antagonists; smallmolecule inhibitors, e.g., methotrexate, leflunomide, sirolimus(rapamycin) and analogs thereof, e.g., CCI-779; Cox-2 and cPLA2inhibitors; NSAIDs; p38 inhibitors; TPL-2; Mk-2; NFkβ inhibitors; RAGEor soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g., small moleculeinhibitors, antibodies thereto, e.g., antibodies to P-selectin);estrogen receptor beta (ERB) agonists or ERB-NFkβ antagonists.

Additionally, the combination treatment useful for administration withthe IL-10 variant molecules or fusion proteins thereof may include TNFinhibitors include, e.g., chimeric, humanized, effectively human, humanor in vitro generated antibodies, or antigen-binding fragments thereof,that bind to TNF; soluble fragments of a TNF receptor, e.g., p55 or p75human TNF receptor or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kDTNF receptor-IgG fusion protein, ENBREL™), p55 kD TNF receptor-IgGfusion protein; and TNF enzyme antagonists, e.g., TNFα converting enzyme(TACE) inhibitors. Other combination treatment with anti-inflammatoryagents/drugs that includes, but not limited to standard non-steroidalanti-inflammatory drugs (NSAIDs) and cyclo-oxygenase-2 inhibitors. NSAIDmay include aspirin, celecoxib, diclofenac, diflunisal, etodolac,ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen,oxaprozin, piroxicam, salsalate, sulindac, and/or tolmetin. Thecyclo-oxygenase-2 inhibitor employed in compositions according to theapplication could, for example, be celecoxib or rofecoxib.

Additional therapeutic agents that can be co-administered and/orco-formulated with IL-10 variant molecules or fusion proteins thereofinclude one or more of: interferon-β, for example, IFN β-1α and IFNβ-1β; COPAXONE®; corticosteroids; IL-1 inhibitors; TNF antagonists(e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNFreceptor or derivatives thereof, e.g., 75 kdTNFR-IgG; antibodies to CD40ligand and CD80; and antagonists of IL-12 and/or IL-23, e.g.,antagonists of a p40 subunit of IL-12 and IL-23 (e.g., inhibitoryantibodies that bind to the p40 subunit of IL-12 and IL-23);methotrexate, leflunomide, and a sirolimus (rapamycin) or an analogthereof, e.g., CCI-779. Other therapeutic agents may include Imfimzi orAtezolizumb.

For purposes of treating NASH, for example, the IL-10 variant moleculesor fusion proteins thereof may be combined with cholesterol loweringagents, such as statins and non-statin drugs. These agents include, butare not limited to simvastatin, atorvastatin, rosuvastatin, lovastatin,pravastatin, gemfibrozil, fluvastatin, cholestyramine, fenofibrate,cholesterol absorption inhibitors, bile acid-binding resins orsequestrants, and/or microsomal triglyceride transfer protein (MTP)inhibitors.

An effective amount of therapeutic will impact the level of inflammationor disease or condition by relieving the symptom. For example, theimpact might include a level of impact that is at least 10%; at least20%; at least about 30%; at least 40%; at least 50%; or more such thatthe disease or condition is alleviated or fully treated.

The pharmaceutical compositions comprising variant IL-10 molecule orfusion proteins thereof is mixed with a pharmaceutically acceptablecarrier or excipient. Various pharmaceutical carriers are known in theart and may be used in the pharmaceutical composition. For example, thecarrier can be any compatible, non-toxic substance suitable fordelivering the variant IL-10 molecule compositions of the application toa patient. Examples of suitable carriers include normal saline, Ringer'ssolution, dextrose solution, and Hank's solution. Carriers may alsoinclude any poloxamers generally known to those of skill in the art,including, but not limited to, those having molecular weights of 2900(L64), 3400 (P65), 4200 (P84), 4600 (P85), 11,400 (F88), 4950 (P103),5900 (P104), 6500 (P105), 14,600 (F108), 5750 (P123), and 12,600 (F127).Carriers may also include emulsifiers, including, but not limited to,polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, toname a few. Non-aqueous carriers such as fixed oils and ethyl oleate mayalso be used. The carrier may also include additives such as substancesthat enhance isotonicity and chemical stability, e.g., buffers andpreservatives, see, e.g., Remington's Pharmaceutical Sciences and U.S.Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa.(1984). Formulations of therapeutic and diagnostic agents may beprepared by mixing with physiologically acceptable carriers, excipients,or stabilizers in the form of lyophilized powders, slurries, aqueoussolutions or suspensions, for example.

Compositions of the application can be administered orally or injectedinto the body. Formulations for oral use can also include compounds tofurther protect the variant IL-10 molecules from proteases in thegastrointestinal tract. Injections are usually intramuscular,subcutaneous, intradermal or intravenous. Alternatively, intra-articularinjection or other routes could be used in appropriate circumstances.Parenterally administered variant IL-10 molecules are preferablyformulated in a unit dosage injectable form (solution, suspension,emulsion) in association with a pharmaceutical carrier and/orpharmaceutically acceptable excipients. In other embodiments,compositions of the application may be introduced into a patient's bodyby implantable or injectable drug delivery system.

Therapeutic Uses of IL-10 Variants

In one embodiment, the present application provides methods of treating,alleviating, or reducing symptoms associated with inflammation,inflammatory disease, or autoimmune disease. The present applicationalso provides for IL-10, IL-10 variant molecules, fusion proteins orchimeric molecules thereof, for use as a medicament for inflammation,inflammatory disease, or autoimmune disease, cancer, or oncology. Thepresent application also contemplates the use of IL-10, IL-10 variantmolecules, fusion proteins or chimeric molecules thereof for use in thetreatment of inflammation or inflammatory disease, or autoimmunedisease, cancer, or oncology. These would include, for example, IBD,Crohn's disease, ulcerative colitis, NASH, NAFLD, hypercholesterolemia,or cancer to name a few. The method contemplates administering atherapeutically effective amount of one or more of the variant IL-10molecule or fusion proteins thereof described herein. In one embodiment,the application includes a method of treating inflammatory diseases orautoimmune diseases comprising administering a therapeutically effectiveamount of a variant IL-10 molecule comprising one or more modificationassociated with the receptor binding domain and/or the regionresponsible for forming the inter-domain angle. In one preferredembodiment, the method includes administering a variant EBV-IL10molecule or fusion proteins thereof. In one preferred embodiment, thevariant IL-10 molecules or fusion proteins thereof useful for treatinginflammatory disease includes variant molecules that have constrainedinter-domain angles, as compared to the wild-type IL-10 molecules and/oralso exhibit lower receptor affinity. In other embodiments, the variantIL-10 molecules or fusion proteins thereof useful for treatinginflammatory disease includes variant molecules that have relaxedinter-domain angles, as compared to the wild-type IL-10 molecules and/oralso exhibit lower receptor affinity. PEGylated forms of the variantIL-10 molecules are also envisioned as part of the present applicationfor inflammatory disease or inflammation.

The inflammatory diseases or autoimmune diseases of the presentapplication include any disease or condition associated with unwanted orundesirable inflammation and immune reaction. These diseases include,but are not limited to, inflammatory bowel disease (IBD), Crohn'sdisease, psoriasis, rheumatoid arthritis, Non-Alcoholic Fatty LiverDisease (NAFLD) or Nonalcoholic steatohepatitis (NASH). In otherembodiments, the diseases or conditions include neurodegenerativedisorders such as Parkinson's disease, amyelotrophic lateral sclerosis(ALS), fatal familial insomnia, Rasmussen's encephalitis, Down'ssyndrome, Huntington's disease, Gerstmann-Straussler-Scheinker disease,tuberous sclerosis, neuronal ceroid lipofuscinosis, subacute sclerosingpanencephalitis, Lyme disease; tse tse's disease (African SleepingSickness), HIV dementia, bovine spongiform encephalopathy (“mad cow”disease); Creutzfeldt Jacob disease; Herpes simplex encephalitis, HerpesZoster cerebellitis, general paresis (syphilis), tuberculous meningitis,tuberculous encephalitis, optic neuritis, granulomatous angiitis,temporal arthritis, cerebral vasculitis, Spatz-Lindenberg's disease,methamphetamine-associated vasculitis, cocaine-associated vasculitis,traumatic brain injury, stroke, Lance-Adams syndrome, post-anoxicencephalopathy, radiation necrosis, limbic encephalitis, Alzheimer'sdisease, progressive supranuclear palsy, striatonigral degeneration,corticocobasal ganglionic degeneration, primary progressive aphasia,frontotemporal dementia associated with chromosome 17, spinal muscularatrophy, HIV-associated myelopathy, HTLV-1-associated myelopathy(Tropical Spastic Paraparesis), tabes dorsalis (syphilis), transversemyelitis, post-polio syndrome, spinal cord injury, radiation myelopathy,Charcot-Marie-Tooth, HIV-associated polyneuropathies,campylobacter-associated motor axonopathies, chronic inflammatorydemyelinating polyneuropathy, diabetic amyotrophy avulsion, phantomlimb, complex regional pain syndrome, diabetic neuropathies,paraneoplastic neuropathies, myotonic dystrophy, HTLV-1-associatedmyopathy, trichinosis, inflammatory myopathies (polymyositis, inclusionbody myositis, dermatomyositis), sickle cell disease,alpha-1-antitrypsin deficiency, tuberculosis, subacute bacterialendocarditis, chronic viral hepatitis, viral cardiomyopathy, Chaga'sdisease, malaria, Coxsackie B infection, macular degeneration, retinitispigmentosa, vasculitis, inflammatory bowel disease, rheumatoidarthritis, bullous pemphigus, Churg-Strauss syndrome, myocardialinfarction, toxic epidermal necrolysis, shock(e.g., acute anaphylacticshock), type-1 diabetes, autoimmune thyroiditis, lymphoma, ovariancancer, Lupus (systemic lupus erythematosus), asthma, progeria,sarcoidosis, type-2 diabetes and metabolic syndrome. Other diseases orconditions associated with inflammation, which are embodiments of theapplication, include inflammatory lung disorders such as bronchitis,oxidant-induced lung injury and chronic obstructive airway disease;inflammatory disorders of the eye including corneal dystrophy, ocularhypertension, trachoma, onchocerciasis, retinitis, uveitis, sympatheticophthalmitis and endophthalmitis; chronic inflammatory disorders of thegum including periodontitis; chronic inflammatory disorders of thejoints including arthritis, septic arthritis and osteoarthritis,tuberculosis arthritis, leprosy arthritis, sarcoid arthritis; disordersof the skin including sclerodermatitis, sunburn, psoriasis and eczema;encephalomyelitis and viral or autoimmune encephalitis; autoimmunediseases including immune-complex vasculitis, and disease of the heartincluding ischemic heart disease, heart failure and cardiomyopathy.Other non-limiting examples of diseases that may benefit from variantIL-10 molecules or fusion proteins thereof include adrenalinsufficiency; hypercholesterolemia; atherosclerosis; bone diseaseassociated with increased bone resorption, e.g., osteoporosis,pre-eclampsia, eclampsia, uremic complications; chronic liver failure,and other disorders associated with inflammation such as cysticfibrosis, tuberculosis, cachexia, ischeimia/reperfusion, hemodialysisrelated conditions, glomerulonephritis, restenosis, inflammatorysequelae of viral infections, hypoxia, hyperbaric oxygen convulsions andtoxicity, dementia, Sydenham's chorea, Huntington's disease, epilepsy,Korsakoffs disease, imbecility related to cerebral vessel disorder, NOmediated cerebral trauma and related sequelae, ischemic brain edema(stroke), migraine, emesis, immune complex disease, allograft rejection,infections caused by invasive microorganisms; and aging.

An IL-10 variant or fusion protein thereof most effective for treatinganti-inflammatory diseases or conditions include those having thelowered capacity of stimulating T-cells. Thus, modifying the receptorbinding domain through an amino acid substitutions at position 75 hasbeen shown by the inventor of the present application to induce theleast amount of T cell stimulation. In particular, EBV IL-10 harboring aA75I substitution in SEQ ID No.: 3 (or SEQ ID No. 57) has been shown todecrease T-cell stimulation (see, e.g., FIG. 8E, denoted as DV06). Thusone particularly preferred embodiment contemplates the use of diabodiesand monobodies with IL-10 variant molecules harboring a DV06 basedmutation (substitution at amino acid position 75 of SEQ ID No.: 3, orSEQ ID No. 57). In a more preferred embodiment, methods of inflammatorydisease will utilize a fusion protein or fusion protein complexcomprising SEQ ID Nos: 26-27; 37; 40;41-42, 43, 48-49 or a combinationthereof.

In another embodiment of the application, the method of treatingincludes administering the IL-10 or variant IL-10 molecule or fusionproteins thereof to treat or reduce symptoms associated with cancer. Themethod contemplates administering a therapeutically effective amount ofone or more of the IL-10 or variant IL-10 molecule or fusion proteinsthereof described herein. In one embodiment, the application includes amethod of treating or reducing symptoms associated with cancercomprising administering a therapeutically effective amount of a variantIL-10 molecule comprising one or more modification associated with thereceptor binding domain and/or the region responsible for forming theinter-domain angle. In one preferred embodiment, the method includesadministering a variant EBV-IL10 molecule or fusion proteins thereof.The variant IL-10 molecules or fusion proteins thereof useful fortreating cancer include variant molecules that have constrainedinter-domain angles, as compared to the wild-type IL-10 molecules and/oralso exhibit higher receptor affinity. The variant IL-10 molecules orfusion proteins thereof useful for treating or reducing symptomsassociated with cancer include variant molecules that have relaxedinter-domain angles, as compared to the wild-type IL-10 molecules and/oralso exhibit higher receptor affinity. PEGylated forms of the variantIL-10 molecules are also envisioned as part of the present applicationfor treating cancer. One particular example of a fusion protein capableof reducing in vivo tumor volume includes an IL-10 variant harboring twosubstitutions at amino acid positions 31 and 75 of SEQ ID No.: 3, whichincludes specific substitutions of V31L and A75I, termed DV07 (e.g., SEQID No.:59). FIGS. 16A-C demonstrates that at varying doses, a fusionprotein comprising the DV07 EBV IL-10 molecule conjugated on a diabodystructure, termed D:DV07, reduced tumor volume over a time course of 7and 10 days. Thus one particularly preferred embodiment contemplates theuse of diabodies and monobodies with IL-10 variant molecules harboring aDV07 based mutation (substitutions at amino acid positions 31 and 75 ofSEQ ID No.: 3; or SEQ ID No: 59). In a more preferred embodiment,methods of treating cancer or oncology or tumors will utilize a fusionprotein or fusion protein complex comprising SEQ ID Nos: 28-29; 33; 34;35-36; 38-39; 46-47, 61, 63, 65, or 67; or a combination thereof.

Cancer or proliferative disorder treatable by the variant IL-10molecules or fusion proteins thereof described herein include variousforms of cancer, including but not limited to, cancer of the uterus,cervix, breast, prostate, testes, penis, gastrointestinal tract, e.g.,esophagus, oropharynx, stomach, small or large intestines, colon, orrectum, kidney, renal cell, bladder, bone, bone marrow, skin, head orneck, skin, liver, gall bladder, heart, lung, pancreas, salivary gland,adrenal gland, thyroid, brain, e.g. gliomas, ganglia, central nervoussystem (CNS) and peripheral nervous system (PNS), and immune system,e.g., spleen or thymus. The present application provides methods oftreating, e.g., immunogenic tumors, non-immunogenic tumors, dormanttumors, virus-induced cancers, e.g., epithelial cell cancers,endothelial cell cancers, squamous cell carcinomas, papillomavirus,adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas,sarcomas, teratocarcinomas, chemically-induced cancers, metastasis, andangiogenesis. The application also contemplates reducing tolerance to atumor cell or cancer cell antigen, e.g., by modulating activity of aregulatory T cell (T_(reg)) and or a CD8⁺ T cell. In a preferredembodiment, the IL-10 variant molecules are particularly useful intreating patients or subjects with liver metastatic disease.

In yet other embodiments, the methods of treating or reducing symptomsassociated with inflammatory disease or cancer include administeringvariant IL-10 molecules or fusion proteins thereof or derivatized formsthereof (e.g., PEGylation) in combination with other therapeutic agents.These therapeutic agents include, without limitation, cytokine orcytokine antagonist, such as IL-12, IL-2, IL-15, interferon-alpha, oranti-epidermal growth factor receptor, doxorubicin, epirubicin, ananti-folate, e.g., methotrexate or fluoruracil, irinotecan,cyclophosphamide, radiotherapy, hormone or anti-hormone therapy, e.g.,androgen, estrogen, anti-estrogen, flutamide, or diethylstilbestrol,surgery, tamoxifen, ifosfamide, mitolactol, an alkylating agent, e.g.,melphalan or cis-platin, etoposide, vinorelbine, vinblastine, vindesine,a glucocorticoid, a histamine receptor antagonist, an angiogenesisinhibitor, radiation, a radiation sensitizer, anthracycline, vincaalkaloid, taxane, e.g., paclitaxel and docetaxel, a cell cycleinhibitor, e.g., a cyclin-dependent kinase inhibitor, a monoclonalantibody against another tumor antigen, a complex of monoclonal antibodyand toxin, a T cell adjuvant, bone marrow transplant, or antigenpresenting cells, e.g., dendritic cell therapy.

In other embodiments, the present application also embodies methods oftreating lipid-related disorders, such as hypercholesterolemia andhypertriglyceridemia, and/or improving lipid parameters such as totalcholesterol, high density lipoprotein (HDL) cholesterol, low densitylipoprotein (LDL) cholesterol, very low density lipoprotein (VLDL)cholesterol, triglycerides, and non-HDL cholesterol, comprisingadministering the variant IL-10 molecules or derivatized form thereof(e.g., PEGylation).

An IL-10 variant or fusion protein thereof most effective for treatinglipid-related diseases or disorders include those having the leastsuppressive capacity on macrophages. Thus, modifying the receptorbinding domain through an amino acid substitutions at position 31(coupled with an increased inter-domain angle of the homodimer) has beenshown by the inventor of the present application to induce the leastamount of macrophage response. In particular, EBV IL-10 harboring a V31Lsubstitution in SEQ ID No.: 3 has been shown to decrease macrophageresponse (see, e.g., FIG. 8A, denoted as DV05, or SEQ ID No. 55). Thusone particularly preferred embodiment contemplates the use of diabodiesand monobodies with IL-10 variant molecules harboring a DV05 basedmutation (substitution at amino acid positions 31 SEQ ID No.: 3, or SEQID No. 55). In a more preferred embodiment, methods of treating lipidbased diseases or disorders will utilize a fusion protein or fusionprotein complex comprising SEQ ID Nos: 24-25; 50-51; or 45.

In yet another embodiment of the application, the IL-10 variantmolecules or fusion proteins thereof, viral IL-10 (including EBV or CMVIL-10), or wild-type IL-10, any of which may optionally include aPEGylation or HESylation, is used in a method of targeting mast cells,by reducing mast cell degranulation. In a preferred embodiment, themethod of targeting mast cells comprise contacting viral IL-10 or IL-10variant molecules to treat seasonal allergies or acute anaphylacticresponse. In another aspect, the IL-10 variant molecules, viral IL-10(including EBV or CMV IL-10), or wild-type IL-10 is used in a method toreduce IgE responsiveness.

In yet another embodiment, the IL-10 variant molecules or fusionproteins thereof of the present application are preferably useful in thedescribed methods (e.g., anti-inflammatory and/or cancer) when thepatient population is screened. In one embodiment, those patients thatexhibit a profile wherein there is an elevated or high IFNγ response aremost susceptible or ideal for use of the IL-10 variant molecules totreat cancer. In another embodiment, those patients that exhibit aprofile wherein there is a decreased or low IFNγ response are mostsusceptible or ideal for use of the IL-10 variant molecules to treatanti-inflammation.

The broad scope of this application is best understood with reference tothe following examples, which are not intended to limit the applicationto any specific embodiments. All citations herein are incorporatedherein by reference to the same extent as if each individual publicationor patent application was specifically and individually indicated to beincorporated by reference.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example, and the application is to be limited by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled; and the application is not to belimited by the specific embodiments that have been presented herein byway of example. Further, all references, patents and patent applicationscited in the foregoing specification are incorporated herein byreference.

EXAMPLES

The following examples are merely illustrative of the variousembodiments of the application and should not be construed in any mannerto limit the scope of the application.

Example 1

EBV-IL-10 variants or fusion proteins thereof are constructed byalteration of the primary sequence through standard molecule biologycloning techniques. The alterations in the primary sequence are designedto alter the affinity of the receptor binding domains as well as open orclose the interdomain angles. The receptor affinity can be altered bychanging amino acids at and around positions 31 and/or 75 in the maturesecreted sequence. The interdomain angles can be altered, for instance,but not limited to, introducing a proline in the non-alpha helicalsequences between helix C and D, and D and E. Prolines drive a kink inthe linear direction of a primary amino acid sequence potentiallyaltering subsequent interdomain angles driven by the secondary andtertiary structures of the D and E helix. Similarly, introduction ofamino acids with bulky side chains such as tryptophan, can introduceless significant alterations to the linear structure of primary aminoacid backbones, resulting in less profound changes to secondary andtertiary structures.

Example 2

The following examples provides a description of how variant IL-10molecules or fusion proteins thereof are assessed in macrophages.

Human blood from healthy patient populations or from patients sufferingfrom an inflammatory disease (e.g., Crohn's disease) are drawn andfreshly drawn Buffy Coats are processed to harvest PBMCs using standardFicoll density gradient centrifugation procedures. PBMCs are thensubjected to enrichment for CD14⁺ monocytes cells using EasySep™ HumanMonocyte Enrichment Kit (Cat #19059, Stem Cell Technologies) andfollowing manufacturer's instructions. The enrichment efficiency areassessed by standard flow cytometry.

The enriched monocytes are plated in 24-well plates at 2×10⁶cells/mL/well in RPMI medium supplemented with 5% human serum and PSG.The cells are treated with serial dilutions (0, 0.1, 1, 10, 100, 1000ng/mL) of the variant IL-10 molecule for 1 hour at 37° C./5% CO2humidified incubator and then exposed to 10 ng/mL LPS (Cat #L4391,Sigma-Aldrich) for 12-16 hours. Following an overnight incubation,supernatants are harvested and inflammatory cytokines (IL-6, TNFα,IL-1β) are measured by either standard ELISA or using iQue Screener(Intellicyt).

In the study, the above described procedure are used to compare theimpacts of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on theimmune suppressive capabilities on macrophages. FIGS. 2A, 2B and 3A, 3Bshow that the EBV-IL10 retained the ability to suppress inflammatorycytokines IL-1β and TNFα, which indicates that despite havingdifferences in inter-domain angle, the EBV-IL10 is capable ofmaintaining its suppressive inflammatory capabilities in a mannersimilar to human IL-10.

Example 3

The following examples provides a description of how variant IL-10molecules or fusion proteins thereof are assessed in human CD8⁺ T-cells.

Human blood from healthy patient populations or from patients sufferingfrom a inflammatory disease (e.g., Crohn's disease) are drawn andfreshly drawn Buffy are processed to harvest PBMCs using standard Ficolldensity gradient centrifugation procedures. The PBMCs are then subjectedto enrichment for CD8⁺ T cells using EasySep™ Human CD8⁺ T CellEnrichment Kit (Cat #19053, Stem Cell Technologies) followingmanufacturer's instructions. The enrichment efficiency are assessed bystandard flow cytometry methods. Enriched cells are suspended in AIMV(Thermo Fisher Scientific, Cat #12055083) culture medium.Twenty-four-well plates are coated with 10 micrograms/mL of anti-CD3(Cat #16-0039-85, Thermo Fisher Scientific) and 2 micrograms/mL ofanti-CD28 (Cat #16-0289-85, Thermo Fisher Scientific) for 2 hours byincubating at 37° C./5% 002 humidified cell culture incubator followedby 1-2 washes with 1× PBS.

The enriched CD8⁺ T cells (3×10⁶/mL/well) are added to theanti-CD³/anti-CD28 coated plates and incubated for 72 hours at 37° C./5%CO2 humidified cell culture incubator.

After 72 hours, the cells are harvested, counted and 100 μl replated inround-bottom 96-well plate (2×105 cells/well) in the presence/absence ofserial dilutions (0, 0.1, 1, 10, 100, 1000 ng/mL—added at 100 μL/well)of the variant IL-10 molecules or a control sample. The testing is runin triplicate. The cells with the variant IL-10 molecules or fusionproteins thereof are incubated for 72 hours at 37° C./5% CO₂ humidifiedincubator. After 72 hours, the cells are collected, washed, replated ina fresh round-bottom 96 well-plate in the presence of soluble ant-CD3(Cat #16-0039-85, Thermo Fisher Scientific) for 4 hours at 37° C./5% CO2humidified incubator.

In the study, the above described procedure are used to compare theimpacts of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on thestimulation of CD8⁺ T cells. FIGS. 2C and 3C show that the EBV-IL10exhibited diminished levels of IFNγ (a measure of T-cells stimulation)when compared to human IL-10. This indicates that by altering theinter-domain angles, there is an ability to modulate stimulation ofT-cells. FIGS. 4A and 4B show that half of the donors treated exhibitthe desired complete anti-inflammatory effects and half do not. Thevariants selected for development will mimic the response of donor 1,complete suppression of inflammatory cytokine secretion in response toLPS by the macrophage cells and the lack of IFNγ induction fromactivated CD8⁺ T cells. Donor 2 exhibited a similar suppression ofinflammatory cytokine secretion by monocytes/macrophages to Donor 1, butonly shifted the curve and maximal activation of T cell IFNγ secretionto the right. IL-10variant molecules that alter receptor affinity andinterdomain angle should further reduce T cell activation in patientssimilar to donor 2.

Example 4

Human monocyctes/macrophages, T cells and murine MC/9 cells purchasedfrom ATCC were cultured as previously stated and the response to mono ordi-N-terminally 5 kDa PEGylated EBV-IL10 was evaluated. PEGylation ofEBV-IL10 results in a slight reduction of macrophage response to LPS(FIG. 5B), but a near complete suppression of IFNγ induction fromstimulated T cells (FIG. 5C). Likewise, PEGylation of EBV-IL10 nearlyabolishes it's stimulatory effect on MC/9 cells (FIG. 5A).

Various forms of EBV-IL-10 variant diabody with anti-CD3α and anti-EGFRVH and VL regions were tested using a MC/9 cell proliferation assay. TheEBV-10 variant portions included D:DV05 (EBV IL-10 with a V31Lmutation), D:DV06 (EBV IL-10 with a A75I mutation), and D:DV07 with V31Land A75I mutations). Additionally a DV07 diabody comprising an anti-HIVand anti-Ebola VH and VL region were also tested. The various variantdiabody forms were compared to human IL-10 and EBV IL-10. Results areprovided in FIG. 15.

Other forms of the EBV-IL-10 fusion proteins were also tested in vitro.In particular DhivDebo:DV06 (SEQ ID Nos: 26 and 27) and DmadcamDebo:DV06(SEQ ID Nos: 41 and 42) were compared to human IL-10 in the macrophageand T-cell response assays described herein. The results are provided inFIGS. 20A and 20B.

Example 5

The following example provides a representative protocol for testing theIL-10 and IL-10 variant molecule and fusion proteins thereof in an invivo tumor model. All in vivo studies are conducted in accordance withthe standard operating procedures and established guidelines approved bythe Institutional Animal Care and Use Committee (“IACUC”).

Eight week-old female Balb/C mice are purchased, quarantined for oneweek, and maintained on normal chow and water with bedding changes 1time per week under a standard 24 hour light/dark cycle.

CT26 tumor cells (2×10⁵) are suspended in Hanks Buffered Salt solutionand subcutaneously implanted into eight week old mice and permitted togrow. CT26 tumor (average 50-150 mm³) bearing wild type Balb/C Envigo),or B cell knockout (Jackson) mice are treated with 0.4 and 0.2 mg/kgthree times a week (q3w), 0.2 and 0.1 mg/kg daily (qd) 5 days with twoday drug holiday, IL-10 or IL-10 variant molecules or fusion proteinsthereof, (e.g., an EBV IL-10 variant molecule harboring two receptorbinding substitutions, DV07 (FIG. 8C), covalently linked to VH and VLfrom two different antibodies or diabody) subcutaneously (scruff) for 10days. The length and width of tumors are measured every three days byelectronic calipers and tumor volume calculated ((L×W²)/2)). B cells inwild type mice are depleted with intravenous (i.v.) administration of200 μg/mouse anti-murine CD20. Results of one such study are provided inFIG. 16, which used a IL-10 variant molecule termed D:DV07, which is aIL-10 variant harboring V31L and A75I mutations comprising variableregions from anti-CD3α and anti-EGFR.

In FIGS. 17A and 17B, two formats of the IL-10 variant fusion proteins(i.e., IL-10 variant comprising both V31L and A75I mutations, DV07)represented by FIGS. 9C (large format) and 9 f (small format) arecompared in an in vivo tumor model. The fusion proteins arenon-targeting fusion proteins and comprising VH and VL regions from ananti-HIV antibody and an anti-ebola antibody (large format) and the VHand VL region from an anti-ebola antibody. The dosing study examined theeffects of the small format non-targeting IL-10 fusion proteinadministered 5 days on, 2 days off (FIG. 17A) compared to pegylatedrecombinant human IL-10 (0.75 mg/kg daily). The dosing study alsoexamined the effects of the large and small formatted non-targetingIL-10 fusion protein administered three times a week (FIG. 17B) comparedto pegylated IL-10 (0.75 mg/kg daily).

Studies using the small and large format IL-10 fusion proteins (i.e.,IL-10 variant comprising both V31L and A75I mutations, DV07) havingtumor targeting capabilities were also tested in vivo. FIG. 18A are theresults from daily administration of various targeting IL-10 variantfusion proteins, where large format (DegfDebo:DV07) and small format(Degf:DV07) were compared to a small format non-targeting (Debo:DV07)IL-10 fusion protein and pegylated IL-10. FIG. 18B are the results fromthree times a week administration of various large format targetingIL-10 variant fusion proteins, where large format (DegfDebo:DV07) atvarious doses (1 mg/kg and 0.25 mg/kg) were compared to a small formatnon-targeting (DhDe:DV07) IL-10 fusion protein and pegylated IL-10. FIG.18C are the results from three times a week administration of varioussmall format targeting IL-10 variant fusion proteins, where small format(Degf:DV07) at various doses (1 mg/kg and 0.25 mg/kg) were compared to asmall format non-targeting (Debo:DV07) IL-10 fusion protein andpegylated IL-10.

Example 6

The following example provides a representative protocol for testing theIL-10 and IL-10 variant molecule and fusion proteins thereof in an invivo cholesterol model. All in vivo studies were conducted in accordancewith the standard operating procedures and established guidelinesapproved by the IACUC.

Eight week-old female C57BL/6J mice are purchased from an appropriatevendor, quarantined for one week and maintained on normal chow and waterwith bedding changes one time/week under a standard 24 hour light/darkcycle.

Eight week-old female C57BL/6J mice Jackson Laboratories are fed a highfat diet (Envigo) for three weeks. Plasma samples are obtained byretro-orbital bleeding of each mouse prior to treatment with an IL-10 orIL-10 variant or fusion protein thereof (e.g., EBV IL-10 variantcomprising a single substitution at amino acid position 31 (V31L) of SEQID No: 3 linked to a diabody (D:DV05 EBV IL-10 variant)). Mice aretreated subcutaneously for two weeks with 0.4 and 0.2 mg/kg three timesa week (q3w) as well as 0.2 and 0.1 mg/kg weekly (qd) 5 days treatmentwith a 2-day therapy holiday. Animals are treated for two weeks andterminal blood draws are taken after which the pre and post-dose plasmacholesterol concentration is quantified. On the day prior to treatmentinitiation, B cells are depleted with intravenous (i.v.) administrationof 200 μg/mouse anti-murine CD20. Results of one such study is providedin FIGS. 19A and 19B.

Example 7

The following example provides a representative protocol for testing theIL-10 and IL-10 variant molecule and fusion proteins thereof in an invivo dextran sodium sulfate (“DSS”) inflammation model. All in vivostudies are conducted in accordance with the standard operatingprocedures and established guidelines approved by the IACUC.

Eight week-old female Balb/C mice are purchased from an appropriatevendor, quarantined for one week and maintained on normal chow and waterwith bedding changes 1 time/week under a standard 24 hour light/darkcycle. B cell knockout (Jackson) mice are fed 4% DSS in water ad libitumfor six days after which they are provided normal water. At day 5, miceare treated with 0.4 and 0.2 mg/kg three times a week (q3w), 0.2 and 0.1mg/kg daily (qd) 5 days with two-day drug holiday, an IL-10 or IL-10variant or fusion protein thereof (e.g., EBV IL-10 variant comprising asingle substitution at amino acid position 75 (A75I) of SEQ ID No: 3linked to a diabody (D:DV06 EBV IL-10 variant)) subcutaneously (scruff)for 10 days. Mice are assessed daily for:

1.) Weight

2.) Stool blood

3.) Gross blood

4.) Stool consistency

Disease activity index are determined by combining scores of;

1. Weight loss

2. Stool consistency

3. Bleeding (divided by 3)

Each score is determined as follows: change in weight (0: <1%, 1: 1-5%,2: 5-10%, 3: 10-15%, 4>15%, stool blood (0: negative, 2: positive) orgross bleeding (4) and stool consistency (0: normal, 2: loose stools, 4:diarrhea).

A LISTING OF PREFERRED EMBODIMENTS

-   1. An Epstein-Barr viral IL-10 (EBV-IL10) variant protein comprising    one or more amino acid additions, deletions, and/or substitutions    exhibiting an altered inter-domain angle and/or an altered affinity    for a cognate receptor when compared to the wild-type EBV-IL10,    wherein the altered inter-domain angle, when dimerized, modulates an    angle of engagement with the cognate receptor.-   2. An EBV-IL10 protein according to the preceding embodiment,    wherein the one or more amino acid additions, deletions, and/or    substitutions is located in the IL-10 receptor binding domain.-   3. An EBV-IL10 protein according to any of the preceding    embodiments, wherein the one or more amino acid additions,    deletions, and/or substitutions is located within alpha helix A    and/or helix D.-   4. An EBV-IL10 protein according to any of the preceding    embodiments, wherein the one or more amino acid additions,    deletions, and/or substitutions resides in the linkage domain of    EBV-IL10.-   5. An EBV-IL10 protein according any of the preceding embodiments,    wherein the one or more amino acid additions, deletions, and/or    substitutions is located within the DE loop of EBV-IL10.-   6. An EBV-IL10 protein according to any of the preceding    embodiments, wherein the one or more amino acid addition, deletion,    and/or substitution is located within the 12 amino acid linker    region found between alpha helix D and alpha helix E or alpha helix    C and alpha helix D, preferably an addition or substitution of a    proline within the 12 amino acid linker region.-   7. An EBV-IL10 protein according to any of the preceding    embodiments, wherein the altered affinity for the cognate receptor    comprises one or more amino acid additions, deletions, and/or    substitutions in the IL-10 receptor binding domain.-   8. An EBV-IL10 protein according to any of the preceding    embodiments, further comprising one or more amino acid additions,    deletions, and/or substitutions located within alpha helix A and/or    alpha helix D.-   9. An EBV-IL10 protein according to any of the preceding    embodiments, further comprising one or more amino acid additions,    deletions, and/or substitutions in the IL-10 receptor binding    domain.-   10. An EBV-IL10 protein according to any of the preceding    embodiments, further comprising one or more amino acid additions,    deletions, and/or substitutions located within alpha helix A and/or    alpha helix D.-   11. An EBV-IL10 protein according to any of the preceding    embodiments, wherein the one or more amino acid additions,    deletions, and/or substitutions is at amino acid position 31 and/or    75 of SEQ ID No. 3.-   12. A monomeric recombinant protein comprising six alpha helices    numbered A-F capable of forming a homodimer with an identical    monomeric protein, wherein alpha helices D and E are linked by an    inter-chain amino acid linker, the linker being modified with an    addition, a deletion, or a substitution of at least one amino acid    that alters an inter-molecular angle of the protein when    homodimerized.-   13. A recombinant protein according to the preceding embodiment,    wherein the protein is a protein from a virus.-   14. A recombinant protein according to any of the preceding    embodiments, wherein the virus is a Epstein-Barr virus (EBV).-   15. A recombinant protein according to any of the preceding    embodiments, wherein the homodimer formed between two identical    monomeric proteins forms an specific angle of interaction with its    cognate receptor.-   16. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction is greater than the    natural wild-type protein.-   17. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction formed upon    homodimerization results in a protein having higher affinity for the    cognate receptor.-   18. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction formed upon    homodimerization results in a protein having a lower affinity for    the cognate receptor.-   19. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction is less than the    natural wild-type protein.-   20. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction results in a protein    having higher affinity for the cognate receptor.-   21. A recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction results in a protein    having less affinity for the cognate receptor.-   22. A recombinant protein according to any of the preceding    embodiments, wherein the monomeric protein is an interleukin 10.-   23. A recombinant protein according to any of the preceding    embodiments, wherein the monomeric protein is an EBV-IL10.-   24. A recombinant protein according to any of the preceding    embodiments, wherein the angle of the protein is imparted by    modifications to the linker resulting in an angle of interaction    with a cognate receptor.-   25. A recombinant variant Epstein-Barr viral IL-10 (EBV-IL10)    protein comprising at least one amino acid addition, deletion, or    substitution to the linker region between alpha helix D and E of    EBV-IL10 and/or to the receptor binding region of EBV-IL10.-   26. An recombinant protein according to the preceding embodiment,    wherein the variant EBV-IL10 protein interacts with an identical    protein resulting in a homodimer having an altered angle of    interaction with its cognate receptor and/or an altered    inter-homodimeric angle.-   27. An recombinant protein according to any of the preceding    embodiments, wherein the variant EBV-IL10 protein forms an angle of    interaction and/or altered inter-homodimeric angle that is greater    than a wild-type EBV-IL10 protein.-   28. An recombinant protein according to any of the preceding    embodiments, wherein the variant EBV-IL10 protein forms an angle of    interaction and/or altered inter-homodimeric angle that is less than    a wild-type EBV-IL10 protein.-   29. An recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction form upon homodimer    formation results in a variant EBV-IL10 protein having increased    affinity to its cognate receptor.-   30. An recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction form upon homodimer    formation results in a variant EBV-IL10 protein having diminished    affinity to its cognate receptor.-   31. An recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction imparts an increase in    affinity to its cognate receptor.-   32. An recombinant protein according to any of the preceding    embodiments, wherein the angle of interaction imparts an decrease in    affinity to its cognate receptor.-   33. An isolated recombinant polynucleotide encoding the protein    according to any of the preceding embodiments.-   34. An isolated recombinant polynucleotide encoding the protein    according to any of the preceding embodiments.-   35. A vector comprising a nucleic acid encoding the protein    according to any of the preceding embodiments.-   36. A host cell comprising the polynucleotide according to any of    the preceding embodiments.-   37. A method of treating or preventing inflammation in a subject    comprising administering to the subject a therapeutically effective    amount of the variant protein according to any of the preceding    embodiments.-   38. A method according to the preceding embodiment, wherein the    altered angle of the variant protein is less than wild-type    EBV-IL10.-   39. A method according to any of the preceding embodiments, wherein    the variant protein binds with moderate affinity to the IL10    receptor when compared to wild-type EBV-IL10.-   40. A method according to any of the preceding embodiments, wherein    the inflammation is Inflammatory Bowel Disease (IBD), Crohn's    disease, Non-Alcoholic Steatohepatiti (NASH), Non-Alcoholic Fatty    Liver Disease (NAFLD), psoriasis, rheumatoid arthritis, acute    anaphylactic shock, and/or seasonal allergies.-   41. A method of treating or preventing auto-immune disease in a    subject comprising administering to the subject a therapeutically    effective amount of the variant protein according to any of the    preceding embodiments.-   42. A method according to the preceding embodiment, wherein the    altered angle of the variant protein is less than wild-type    EBV-IL10.-   43. A method according to any of the preceding embodiments, wherein    the variant protein binds with moderate affinity to the IL10    receptor when compared to wild-type EBV-IL10.-   44. A method of treating or preventing IBD or Crohn' s Disease in a    subject comprising administering to the subject a therapeutically    effective amount of the variant protein according to any of the    preceding embodiments.-   45. A method according to the preceding embodiment, wherein the    altered angle of the variant protein is less than wild-type    EBV-IL10.-   46. A method according to any of the preceding embodiments, wherein    the variant protein binds with moderate affinity to the IL10    receptor when compared to wild-type EBV-IL10.-   47. A method of treating or preventing Non-Alcoholic Fatty Liver    Disease (NAFLD) or Non-Alcoholic Steatohepatiti (NASH) in a subject    comprising administering to the subject a therapeutically effective    amount of the variant protein according to claim 1.-   48. A method according to the preceding embodiment, wherein the    altered angle of the variant protein is less than wild-type    EBV-IL10.-   49. A method according to any of the preceding embodiments, wherein    the variant protein binds with moderate affinity to the IL10    receptor when compared to wild-type EBV-IL10.-   50. A method of treating or preventing cancer in a subject    comprising administering to the subject a therapeutically effective    amount of the variant protein according to any of the preceding    embodiments.-   51. A method according to the preceding embodiment, wherein the    altered angle of the variant protein is greater than wild-type    EBV-IL10.-   52. A method according to any of the preceding embodiments, wherein    the variant protein binds with increased affinity to the IL10    receptor when compared to wild-type EBV-IL10.-   53. An engineered fusion protein comprising at least one monomer of    IL-10 or IL-10 variant molecule conjugated at a first terminal end    of the fusion protein, at least one cytokine or monomer thereof    conjugated at a second terminal end of the fusion protein, and a    linker or spacer, wherein the linker or spacer connects the first    and second terminal ends.-   54. A fusion protein according to the preceding embodiment, wherein    the linker or spacer is a constant region of an antibody.-   55. A fusion protein according to any of the preceding embodiments,    wherein the constant region is derived from an IgG1, IgG2, IgG3,    IgG4, IgA, IgM, IgD, or IgE.-   56. A fusion protein according to any of the preceding embodiments,    wherein the linker or spacer further comprises at least two    interchain disulfide bonds.-   57. A fusion protein according to any of the preceding embodiments,    wherein the linker or spacer is a scFv, a diabody, or fragments    thereof.-   58. A fusion protein according to any of the preceding embodiments,    wherein the constant region is a heavy chain constant (CH) region 1,    CH2, CH3, or any combination thereof.-   59. A fusion protein according to any of the preceding embodiments,    wherein the at least one IL-10 or IL-10 variant molecule is    conjugated at the fusion protein's N-terminal end, the C-terminal    end, or both.-   60. A fusion protein according to any of the preceding embodiments,    wherein the at least one cytokine conjugated at another terminal end    includes IL-10, IL-10 variant molecule IL-6, IL-4, IL-1, IL-2, IL-3,    IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF,    G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis factors -α,    -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination    thereof.-   61. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises two IL-10 or IL-10 variant    molecules conjugated at the N-terminal end of the fusion protein and    two IL-10 or IL-10 variant molecules conjugated at the C-terminal    end of the fusion protein.-   62. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises two IL-10 or IL-10 variant    molecules conjugated at the N-terminal end of the fusion protein and    at least one IL-2 molecules conjugated at the C-terminal end of the    fusion protein.-   63. A fusion protein according to any of the preceding embodiments,    wherein the C-terminal further comprises an IL-6, IL-4, IL-1, IL-2,    IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29,    GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis    factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.-   64. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises two IL-10 or IL-10 variant    molecules conjugated at the N-terminal end of the fusion protein and    at least one IL-15 molecules conjugated at the C-terminal end of the    fusion protein.-   65. A fusion protein v, wherein the C-terminal further comprises an    IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26,    IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β,    or tumor necrosis factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11,    or IL-13.-   66. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises two IL-10 or IL-10 variant    molecules conjugated at the N-terminal end of the fusion protein and    at least one IL-2 molecules conjugated at the C-terminal end of the    fusion protein.-   67. A fusion protein according to any of the preceding embodiments,    wherein the C-terminal further comprises an IL-10, IL-10 variant    molecule IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9,    IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α,    -β, -γ, TGF-β, or tumor necrosis factors -α, -β, basic FGF, EGF,    PDGF, IL-4, IL-11, or IL-13.-   68. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein is fabricated on a single chain variable    fragment (scFv) scaffold.-   69. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein is fabricated on a diabody scaffold.-   70. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein is fabricated on an Fab scaffold.-   71. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein complexes with another fusion protein    having at least one monomer of IL-10 or IL-10 variant molecule    conjugated at a first terminal end of the fusion protein, at least    one cytokine or monomer thereof conjugated at a second terminal end    of the fusion protein, and a linker or spacer, wherein the linker or    spacer connects the first and second terminal ends.-   72. A method of treating cancer in a subject in need thereof    comprising administering to the subject the engineered fusion    protein according to any of the preceding embodiments.-   73. A method of treating or preventing IBD or Crohn's Disease    comprising administering to a subject the engineered fusion protein    according to any of the preceding embodiments.-   74. A method of treating or preventing Non-Alcoholic Fatty Liver    Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH) in a subject    comprising administering to the subject the engineered fusion    protein according to any of the preceding embodiments.-   75. A method of activating CD8 positive T-cell comprising    administering the engineered fusion protein according to any of the    preceding embodiments.-   76. A method according to any of the preceding embodiments, wherein    the administration is in vitro administration.-   77. A method according to any of the preceding embodiments, wherein    the administration is in vivo administration to a subject in need    thereof, wherein the subject has been diagnosed with cancer, IBD or    Crohn's disease, or NAFLD or NASH.-   78. A method according to any of the preceding embodiments, wherein    the fusion protein comprises a IL-10 or IL-10 variant molecule at    the first terminal end of the fusion protein and an IL-2 and/or    IL-15 at the second terminal end of fusion protein.-   79. A method of treating cancer in a subject in need thereof    comprising administering to the subject a bispecific T-cell engager    (BITE) and an IL-10, an IL-10 variant molecule, or an engineered    fusion protein comprising an IL-10 or an IL-10 variant molecule.-   80. A method according to the preceding embodiment, wherein the    engineered fusion protein comprises at least one IL-10 or IL-10    variant molecule conjugated at a first terminal end of the fusion    protein, at least one cytokine conjugated at a second terminal end    of the fusion protein, and a linker or spacer, wherein the linker or    spacer connects the first and second terminal ends.-   81. A method according to any of the preceding embodiments, wherein    the IL-10, IL-10 variant molecule, or the engineered fusion protein    comprising an IL-10 or an IL-10 variant molecule increases and    sustains T-cell receptor complex (CD3) signal transduction.-   82. A method of treating or preventing inflammation in a subject    comprising administering to the subject a therapeutically effective    an amount of a nucleotide sequence encoding a variant IL-10    molecule.-   83. A method according to the preceding embodiment, wherein the    nucleotide sequence is DNA, RNA, or modified variants thereof.-   84. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is a mRNA or a modified mRNA linked to a    nucleoside.-   85. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is capable of in vivo expressing the variant    IL-10 molecule within a cell, tissue, or organism.-   86. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is delivered to a cell, tissue, or organism    by a cell penetrating peptide, a hydrophobic moiety, an    electrostatic complex, a liposome, a ligand, a liposomal    nanoparticle, a lipoprotein (preferably HDL or LDL), a folate    targeted liposome, an antibody (such as Folate receptor, transferrin    receptor), a targeting peptide, or by an aptamer.-   87. A method of treating or preventing auto-immune disease in a    subject comprising administering to the subject a therapeutically    effective amount of a nucleotide sequence encoding a variant IL-10    molecules.-   88. A method according to the preceding embodiment, wherein the    nucleotide sequence is DNA, RNA, or modified variants thereof.-   89. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is a mRNA or a modified mRNA linked to a    nucleoside.-   90. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is capable of in vivo expressing the variant    IL-10 molecule within a cell, tissue, or organism.-   91. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is delivered to a cell, tissue, or organism    by a cell penetrating peptide, a hydrophobic moiety, an    electrostatic complex, a liposome, a ligand, a liposomal    nanoparticle, a lipoprotein (preferably HDL or LDL), a folate    targeted liposome, an antibody (such as Folate receptor, transferrin    receptor), a targeting peptide, or by an aptamer.-   92. A method of treating or preventing IBD or Crohn' s Disease in a    subject comprising administering to the subject a therapeutically    effective amount of a nucleotide sequence encoding a variant IL-10    molecule.-   93. A method according to the preceding embodiment, wherein the    nucleotide sequence is DNA, RNA, or modified variants thereof.-   94. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is a mRNA or a modified mRNA linked to a    nucleoside.-   95. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is capable of in vivo expressing the variant    IL-10 molecule within a cell, tissue, or organism.-   96. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is delivered to a cell, tissue, or organism    by a cell penetrating peptide, a hydrophobic moiety, an    electrostatic complex, a liposome, a ligand, a liposomal    nanoparticle, a lipoprotein (preferably HDL or LDL), a folate    targeted liposome, an antibody (such as Folate receptor, transferrin    receptor), a targeting peptide, or by an aptamer.-   97. A method of treating or preventing Non-Alcoholic Fatty Liver    Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH) in a subject    comprising administering to the subject a therapeutically effective    amount of a nucleotide sequence encoding a variant IL-10 molecule.-   98. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is DNA, RNA, or modified variants thereof.-   99. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is a mRNA or a modified mRNA linked to a    nucleoside.-   100. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is capable of in vivo expressing the variant    IL-10 molecule within a cell, tissue, or organism.-   101. A method according to any of the preceding embodiments, wherein    the nucleotide sequence is delivered to a cell, tissue, or organism    by a cell penetrating peptide, a hydrophobic moiety, an    electrostatic complex, a liposome, a ligand, a liposomal    nanoparticle, a lipoprotein (preferably HDL or LDL), a folate    targeted liposome, an antibody (such as Folate receptor, transferrin    receptor), a targeting peptide, or by an aptamer.-   102. A fusion protein comprising a monomeric IL-10 molecule or a    variant thereof linked to two variable regions from at least two    different antibodies, wherein the two variable regions are    configured as a heavy chain variable (VH) region from a first    antibody linked to a light chain variable (VL) region from a second    antibody or a VL from the first antibody linked to a VH from the    second antibody.-   103. A fusion protein according to any of the preceding embodiments,    wherein the monomeric IL-10 molecule or a variant thereof includes    at least one amino acid substitution that increases or decreases    affinity to an IL-10 receptor.-   104. A fusion protein according to any of the preceding embodiments,    wherein the monomeric IL-10 molecule or a variant thereof includes    at least one amino acid substitution that increases affinity to an    IL-10 receptor.-   105. A fusion protein according to any of the preceding embodiments,    wherein the monomeric IL-10 molecule or a variant thereof is an    Epstein Barr virus (EBV) IL-10 homolog of SEQ ID No.: 3.-   106. A fusion protein according to any of the preceding embodiments,    wherein the EBV IL-10 homolog includes an amino acid substitution at    position 31, 75, or both.-   107. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 31.-   108. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 75.-   109. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    positions 31 and 75.-   110. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an V31L amino acid    substitution.-   111. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an A75I amino acid    substitution.-   112. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an V31L and A75I amino acid    substitution.-   113. A fusion protein according to any of the preceding embodiments,    wherein the VH region of the first antibody is from an anti-HIV    monoclonal antibody and the VL region of the second antibody is from    an anti-ebola monoclonal antibody.-   114. A fusion protein according to any of the preceding embodiments,    wherein the VH region of the first antibody is from an anti-ebola    monoclonal antibody and the VL region of the second antibody is from    an anti-HIV monoclonal antibody.-   115. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein is an amino acid sequence selected from    SEQ ID Nos.: 24-28, 29, 33-51, 61, 63, 65, or 67.-   116. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein is a diabody.-   117. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises a configuration selected    from: (a) a VH region of the first antibody linked at its carboxy    terminal end to an amino terminal end of a VL region of the second    antibody subsequently linked to an amino terminal end of a monomer    of IL-10 or a variant thereof; or (b) an IL-10 molecule or a variant    thereof linked at its carboxy terminal end to an amino terminal end    of a VH region of the second antibody subsequently linked to an    amino terminal end of a VL region of the first antibody.-   118. A fusion protein according to any of the preceding embodiments,    wherein configuration (a) and (b) together form a diabody complex.-   119. A fusion protein according to any of the preceding embodiments,    further comprising a linker between the VH region and the VL region.-   120. A fusion protein according to any of the preceding embodiments,    wherein the amino acid sequence is selected from SEQ ID Nos: 24-28,    29, 33-53, 61, 63, 65, 67.-   121. A fusion protein according to any of the preceding embodiments,    wherein the first and second antibodies comprises one or more amino    acid substitutions that reduce antigenicity in a subject.-   122. An immunoconjugate complex comprising i) a first fusion protein    comprising at its amino terminal end a heavy chain variable region    (VH) of a first antibody linked to a light chain variable region    (VL) of a second antibody further linked to a monomer of IL-10 or    variant thereof; and ii) a second fusion protein comprising at its    amino-terminal end a monomer of IL-10 or variant thereof linked to a    VH of the second antibody further linked to a VL of the first    antibody, wherein the VH and VL of the first and second antibodies    associate into a diabody and the monomers of IL-10 form a functional    dimeric IL-10 molecule.-   123. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 includes at least one    amino acid substitution that increases or decreases affinity to an    IL-10 receptor.-   124. An immunoconjugate complex according to any of the preceding    embodiments, wherein the IL-10 molecule includes at least one amino    acid substitution that increases affinity to an IL-10 receptor.-   125. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 is an Epstein Barr virus    (EBV) IL-10 homolog of SEQ ID No. 3.-   126. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV IL-10 homolog includes an amino acid    substitution at position 31, 75, or both.-   127. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at position 31.-   128. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at position 75.-   129. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at positions 31 and 75.-   130. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an V31L amino    acid substitution.-   131. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an A75I amino    acid substitution.-   132. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an V31L and A75I    amino acid substitution.-   133. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first antibody is from an anti-HIV    monoclonal antibody and the second antibody is from an anti-ebola    monoclonal antibody.-   134. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first fusion protein is an amino acid    sequence selected from SEQ ID Nos.: 24, 26, 28, 35, 38, 41, 46, 48,    or 50.-   135. An immunoconjugate complex according to any of the preceding    embodiments, wherein the second fusion protein is an amino acid    sequence selected from SEQ ID Nos.: 25,27, 29, 36, 39, 42, 47, 49,    or 51.-   136. An immunoconjugate complex according to any of the preceding    embodiments, further comprising a linker between the VH regions and    the VL regions.-   137. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 in the first fusion    protein is linked to the VL region by its amino terminal end.-   138. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 in the second fusion    protein is linked to the VH region by its carboxy terminal end.-   139. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first and second antibodies comprises one    or more amino acid substitutions that reduce antigenicity in a    subject.-   140. A diabody comprising a first peptide chain comprising a heavy    chain variable region (VH) from a first antibody, a light chain    variable region (VL) from a second antibody, and a monomeric IL-10;    and a second peptide chain comprising a VH and a VL from a second    antibody and a monomeric IL-10 molecule, wherein the VH region of    the first antibody associates with the VL region of first antibody    and the VH region of the second antibody associates with the VL    region of second antibody thereby allowing the monomeric IL-10    molecules on each peptide chain to form a functional IL-10 dimer.-   141. A diabody according to any of the preceding embodiments,    wherein the monomeric IL-10 includes at least one amino acid    substitution that increases or decreases affinity to an IL-10    receptor.-   142. A diabody according to any of the preceding embodiments,    wherein the monomeric IL-10 molecule includes at least one amino    acid substitution that increases affinity to an IL-10 receptor.-   143. A diabody according to any of the preceding embodiments,    wherein the monomeric IL-10 molecule is an Epstein Barr virus (EBV)    IL-10 homolog of SEQ ID No. 3.-   144. A diabody according to any of the preceding embodiments,    wherein the EBV IL-10 homolog includes an amino acid substitution at    position31, 75, or both.-   145. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 31.-   146. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 75.-   147. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    positions 31 and 75.-   148. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an V31L amino acid    substitution.-   149. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an A75I amino acid    substitution.-   150. A diabody according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an V31L and A75I amino acid    substitution.-   151. A diabody according to any of the preceding embodiments,    wherein the first antibody is from an anti-HIV monoclonal antibody    and the second antibody is from an anti-ebola monoclonal antibody.-   152. A diabody according to any of the preceding embodiments,    wherein the first peptide chain is an amino acid sequence selected    from SEQ ID Nos.: 24, 26, 28, 35, 38, 41, 46, 48, or 50.-   153. A diabody according to any of the preceding embodiments,    wherein the second peptide chain is an amino acid sequence selected    from SEQ ID Nos.: 25,27, 29, 36, 39, 42-47, 49, or 51.-   154. A diabody according to any of the preceding embodiments,    further comprising a linker between the VH regions and the VL    regions.-   155. A diabody according to any of the preceding embodiments,    wherein the first and second antibodies comprises one or more amino    acid substitutions that reduce antigenicity in a subject.-   156. A diabody according to any of the preceding embodiments,    wherein the monomer of IL-10 is linked to the first and second    peptide chain by its carboxy terminal end.-   157. A diabody according to any of the preceding embodiments,    wherein the first and second antibodies comprises one or more amino    acid substitutions that reduce antigenicity in a subject.-   158. An immunoconjugate complex comprising i) a first fusion protein    comprising at its amino terminal end a heavy chain variable (VH)    region of a first antibody and a monomeric IL-10 molecule linked to    by its amino terminal end; and ii) a second fusion protein    comprising at its amino terminal end a monomer of IL-10 linked to a    light chain variable region (VL) of the first antibody, wherein the    VH region of the first antibody associates with the VL region of    first antibody thereby allowing the monomeric IL-10 molecules on    each peptide chain to form a functional IL-10 dimer.-   159. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 includes at least one    amino acid substitution that increases or decreases affinity to an    IL-10 receptor.-   160. An immunoconjugate complex according to any of the preceding    embodiments, wherein the IL-10 molecule includes at least one amino    acid substitution that increases affinity to an IL-10 receptor.-   161. An immunoconjugate complex according to any of the preceding    embodiments, wherein the monomer of IL-10 is an Epstein Barr virus    (EBV) IL-10 homolog of SEQ ID No. 3.-   162. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV IL-10 homolog includes an amino acid    substitution at position 31, 75, or both.-   163. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at position 31.-   164. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at position 75-   165. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an amino acid    substitution at positions 31 and 75.-   166. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an V31L amino    acid substitution.-   167. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an A75I amino    acid substitution.-   168. An immunoconjugate complex according to any of the preceding    embodiments, wherein the EBV-IL-10 homolog includes an V31L and A75I    amino acid substitution.-   169. An immunoconjugate complex according to any of the preceding    embodiments, wherein the VH and VL regions of the first antibody are    from an anti-epidermal growth factor receptor (EGFR) monoclonal    antibody.-   170. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first fusion protein further comprises a VL    region from a second antibody linking the VH region of the first    antibody to the monomeric IL-10 and wherein the second fusion    protein further comprises a VH region from the second antibody    linking the monomeric IL-10 to the VL region.-   171. An immunoconjugate complex according to any of the preceding    embodiments, wherein the VH and VL regions of the second antibody    are from an anti-Ebola monoclonal antibody.-   172. An immunoconjugate complex according to any of the preceding    embodiments, wherein the variable regions are linked to the monomers    of IL-10 through linkers.-   173. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first and second antibodies comprises one    or more amino acid substitutions that reduce antigenicity in a    subject.-   174. An immunoconjugate complex according to any of the preceding    embodiments, wherein the first and second antibodies comprises one    or more amino acid substitutions that reduce antigenicity in a    subject.-   175. A fusion protein comprising variable light (VL) and variable    heavy (VH) regions of a first antibody fused to monomers of IL-10,    wherein the IL-10 monomers are directly linked to one another.-   176. A fusion protein according to any of the preceding embodiments,    wherein the IL-10 monomers are linked from a carboxy terminal end of    a first IL-10 monomer to an amino terminal end of a second IL-10    monomer.-   177. A fusion protein according to any of the preceding embodiments,    wherein the fusion protein comprises the following configuration in    amino to carboxy terminal fashion: the VL region of the first    antibody is linked to a first IL-10 monomer, linked to a second    IL-10 monomer, linked to the VH region of the first antibody.-   178. A fusion protein according to any of the preceding embodiments,    further comprising a VH region of a second antibody linked to the    amino terminal end of the VL region of the first antibody and a VL    region of a second antibody linked to the carboxy terminal end of    the VH region of the first antibody.-   179. A fusion protein according to any of the preceding embodiments,    wherein the IL-10 monomers each include at least one amino acid    substitution that increases or decreases affinity to an IL-10    receptor.-   180. A fusion protein according to any of the preceding embodiments,    wherein the IL-10 monomers each include at least one amino acid    substitution that increases affinity to an IL-10 receptor.-   181. A fusion protein according to any of the preceding embodiments,    wherein the IL-10 monomer is an Epstein Barr virus (EBV) IL-10    homolog of SEQ ID No.: 3.-   182. A fusion protein according to claim 181, wherein the EBV IL-10    homolog includes an amino acid substitution at position 31, 75, or    both.-   183. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 31.-   184. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an amino acid substitution at    position 75.-   185. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an V31L amino acid    substitution.-   186. A fusion protein according to any of the preceding embodiments,    wherein the EBV-IL-10 homolog includes an A75I amino acid    substitution.-   187. A fusion protein according to any of the preceding embodiments,    wherein the first antibody is an anti-Ebola monoclonal antibody.-   188. A fusion protein according to any of the preceding embodiments,    wherein the first antibody is an anti-epidermal growth factor    receptor (EGFR) monoclonal antibody.-   189. A fusion protein according to any of the preceding embodiments,    wherein the first antibody is an anti-Ebola monoclonal antibody and    the second antibody is an anti-EGFR monoclonal antibody.-   190. A method of treating a disease, disorder, or condition in a    patient in need thereof, comprising administering to the patient a    therapeutically effective amount of an Epstein Barr virus (EBV)    IL-10 immunoconjugate complex, wherein the immunoconjugate complex    has a molecular weight of about 60 to 155 kDa, wherein the    therapeutically effective amount is in the range of about 0.5    microgram/kilogram to 100 micrograms/kilogram, and wherein the EBV    IL-10 portion of the immunoconjugate is derived from SEQ ID No.: 3.-   191. A method according to any of the preceding embodiments, wherein    the immunoconjugate complex is administered monthly, bimonthly,    weekly, twice a week, three times a week, or daily.-   192. A method according to any of the preceding embodiments, wherein    the variant EBV IL-10 is a variant comprising at least one amino    acid substitution that increases or decreases binding to the IL-10    receptor-   193. A method according to any of the preceding embodiments, wherein    the variant EBV IL-10 comprises an amino acid substitution at    positions 31, 75 or both of SEQ ID No.: 3.-   194. A method according to any of the preceding embodiments, wherein    the variant EBV IL-10 comprises an V31L amino acid substitution.-   195. A method according to any of the preceding embodiments, wherein    the EBV IL-10 comprises an A75I amino acid substitution.-   196. A method according to any of the preceding embodiments, wherein    the EBV IL-10 comprises V31L and A75I amino acid substitutions.-   197. A method according to any of the preceding embodiments, wherein    the immunoconjugate complex is a complex of two fusion proteins.-   198. A method according to any of the preceding embodiments, wherein    the immunoconjugate complex comprises i. a first fusion protein    comprising at its amino-terminal end a heavy chain variable region    (VH) of a first antibody linked to a light chain variable region    (VL) of a second antibody further linked to a carboxy terminal end    of a monomer of EBV IL-10; and ii. a second fusion protein    comprising at its amino-terminal end a monomer of EBV IL-10 linked    to a VH of the second antibody further linked to a VL of the first    antibody, wherein the VHs and VLs of the first and second antibodies    associate into a diabody and the monomers of EBV IL-10 form a    functional dimeric EBV IL-10 molecule.-   199. A method according to any of the preceding embodiments, wherein    the first antibody and second antibody are different antibodies.-   200. A method according to any of the preceding embodiments, wherein    the first antibody is an anti-HIV monoclonal antibody and the second    antibody is an anti-ebola monoclonal antibody.-   201. A method according to claim 202, wherein the fusion protein is    an amino acid sequence selected from SEQ ID Nos: 24-51.-   202. A method according to claim 202, wherein the first fusion    protein is an amino acid sequence selected from SEQ ID Nos.: 24, 26,    28, 35, 38, 41, 46, 48, or 50.-   203. A method according to claim 202, wherein the second fusion    protein is an amino acid sequence selected from SEQ ID Nos.: 25,27,    29, 36, 39, 42, 47, 49, or 51.-   204. A method according to any of the preceding embodiments, wherein    the immunoconjugate complex is a diabody comprising EBV IL-10    monomers fused on either terminal ends, wherein the EBV IL-10    monomers are capable of associating into a functional EBV IL-10    dimer.-   205. A method according to any of the preceding embodiments, wherein    the disease, disorder, or condition is selected from cancer,    inflammatory disease, autoimmune disease, or cholesterol.-   206. A method according to any of the preceding embodiments, wherein    the EBV IL-10 immunoconjugate complex is administered in an amount    sufficient to a maintain steady IL-10 serum concentration based on    administering at least every 2 to 3 days.-   207. A method according to any of the preceding embodiments, wherein    the immunoconjugate is capable of suppressing TNFα secretion and    inducing IFNγ production at similar concentrations to wild type    IL-10.-   208. A method according to any of the preceding embodiments, wherein    the EBV IL-10 immunoconjugate complex has similar activity to    wild-type IL-10.-   209. A method according to any of the preceding embodiments, wherein    the immunoconjugate complex comprises-   (a) VH region of the first antibody at the N-terminal end linked to    a VL region of the second antibody linked to a carboxy terminus of    an IL-10 molecule; and-   (b) an IL-10 molecule linked to a VH region of the second antibody    linked to a VL region of the first antibody.-   210. A method of treating cancer in a patient in need thereof,    comprising administering to the patient a diabody comprising a first    peptide chain having a heavy chain variable region (VH) from a first    antibody, a light chain variable region (VL) from a second antibody,    and a monomeric IL-10 molecule; and a second peptide chain having a    VH and a VL from a second antibody and a monomeric IL-10 molecule,    wherein the VH region of the first antibody associates with the VL    region of first antibody and the VH region of the second antibody    associates with the VL region of second antibody thereby allowing    the monomeric IL-10 molecules on each peptide chain to form a    functional IL-10 dimer.-   211. A method according to any of the preceding embodiments, wherein    the monomeric IL-10 is an Epstein Barr virus (EBV) IL-10 homolog of    SEQ ID No.: 3.-   212. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an amino acid substitution at position 31 of    SEQ ID No.: 3.-   213. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an V31L amino acid substitution.-   214. A method according to any of the preceding embodiments, wherein    the VH region of the first antibody is from an anti-HIV monoclonal    antibody and the VL region of the second antibody is from an    anti-ebola monoclonal antibody.-   215. A method according to any of the preceding embodiments, wherein    the first peptide chain is an amino acid sequence selected from SEQ    ID No.: 28, 35, 38, 46.-   216. A method according to any of the preceding embodiments, wherein    the second peptide chain is an amino acid sequence selected from SEQ    ID Nos.: 29, 36, 39, 47.-   217. A method according to any of the preceding embodiments, further    comprising a linker between the VH regions and the VL regions.-   218. A method according to any of the preceding embodiments, wherein    VH and VL each comprise one or more amino acid substitutions that    reduce antigenicity in the patient.-   219. A method according to any of the preceding embodiments, wherein    the first antibody is from an anti-HIV monoclonal antibody and the    second antibody is from an anti-ebola monoclonal antibody.-   220. A method according to any of the preceding embodiments, wherein    the diabody is formed with two peptide chains with the following    amino to carboxy terminus configurations: (a) a first peptide    comprising a VH region of the first antibody linked to a VL region    of the second antibody that is then linked to a amino terminus of an    IL-10 monomer or variant thereof; and (b) a second peptide    comprising an IL-10 monomer linked to a VH region of the second    antibody that is then linked to a VL region of the first antibody.-   221. A method of treating cholesterol in a patient in need thereof,    comprising administering to the patient a cholesterol reducing    amount of a diabody comprising a first peptide chain having a heavy    chain variable region (VH) from a first antibody, a light chain    variable region (VL) from a second antibody, and a monomeric IL-10;    and a second peptide chain having a VH and a VL from a second    antibody and a monomeric IL-10 molecule, wherein the VH region of    the first antibody associates with the VL region of first antibody    and the VH region of the second antibody associates with the VL    region of second antibody thereby allowing the monomeric IL-10    molecules on each peptide chain to form a functional IL-10 dimer.-   222. A method according to any of the preceding embodiments, wherein    the monomeric IL-10 is an Epstein Barr virus (EBV) IL-10 homolog of    SEQ ID No.: 3.-   223. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an amino acid substitution at position 31 of    SEQ ID No.: 3.-   224. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an V31L amino acid substitution.-   225. A method according to any of the preceding embodiments, wherein    the VH region of the first antibody is from an anti-HIV monoclonal    antibody and the VL region of the second antibody is from an    anti-ebola monoclonal antibody.-   226. A method according to any of the preceding embodiments, wherein    the first peptide chain is an amino acid sequence selected from SEQ    ID No.: 24 or 50.-   227. A method according to any of the preceding embodiments, wherein    the second peptide chain is an amino acid sequence selected from SEQ    ID No.: 25 or 51.-   228. A method according to any of the preceding embodiments, further    comprising a linker between the VH regions and the VL regions.-   229. A method according to any of the preceding embodiments, wherein    VH and VL each comprise one or more amino acid substitutions that    reduce antigenicity in the patient.-   230. A method according to any of the preceding embodiments, wherein    the first antibody is from an anti-HIV monoclonal antibody and the    second antibody is from an anti-ebola monoclonal antibody.-   231. A method according to any of the preceding embodiments, wherein    the diabody is formed with two peptide chains with the following    amino to carboxy terminus configurations: (a) a first peptide    comprising a VH region of the first antibody linked to a VL region    of the second antibody that is then linked to a amino terminus of an    IL-10 monomer or variant thereof; and (b) a second peptide    comprising an IL-10 monomer or variant thereof linked to a VH region    of the second antibody that is then linked to a VL region of the    first antibody.-   232. A method of treating nonalcoholic steatohepatitis (NASH) or    nonalcoholic fatty liver disease (NAFLD) in a patient in need    thereof, comprising administering to the patient an NASH or NAFLD    amount of a diabody comprising a first peptide chain having a heavy    chain variable region (VH) from a first antibody, a light chain    variable region (VL) from a second antibody, and a monomeric IL-10;    and a second peptide chain having a VH and a VL from a second    antibody and a monomeric IL-10 molecule, wherein the VH region of    the first antibody associates with the VL region of first antibody    and the VH region of the second antibody associates with the VL    region of second antibody thereby allowing the monomeric IL-10    molecules on each peptide chain to form a functional IL-10 dimer.-   233. A method according to any of the preceding embodiments, wherein    the monomeric IL-10 is an Epstein Barr virus (EBV) IL-10 homolog of    SEQ ID No.: 3.-   234. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an amino acid substitution at position 31 of    SEQ ID No.: 3.-   235. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an V31L amino acid substitution.-   236. A method according to any of the preceding embodiments, wherein    the VH region of the first antibody is from an anti-HIV monoclonal    antibody and the VL region of the second antibody is from an    anti-ebola monoclonal antibody.-   237. A method according to any of the preceding embodiments, wherein    the first peptide chain is an amino acid sequence selected from SEQ    ID Nos.: 24 or 50.-   238. A method according to any of the preceding embodiments, wherein    the second peptide chain is an amino acid sequence selected from SEQ    ID Nos.: 25 or 51.-   239. A method according to any of the preceding embodiments, further    comprising a linker between the VH regions and the VL regions.-   240. A method according to any of the preceding embodiments, wherein    VH and VL each comprise one or more amino acid substitutions that    reduce antigenicity in the patient.-   241. A method according to any of the preceding embodiments, wherein    the first antibody is from an anti-HIV monoclonal antibody and the    second antibody is from an anti-ebola monoclonal antibody.-   242. A method of treating inflammation in a patient in need thereof,    comprising administering to the patient an anti-inflammatory amount    of a diabody comprising a first peptide chain having a heavy chain    variable region (VH) from a first antibody, a light chain variable    region (VL) from a second antibody, and a monomeric IL-10; and a    second peptide chain having a VH and a VL from a second antibody and    a monomeric IL-10 molecule, wherein the VH region of the first    antibody associates with the VL region of first antibody and the VH    region of the second antibody associates with the VL region of    second antibody thereby allowing the monomeric IL-10 molecules on    each peptide chain to form a functional IL-10 dimer.-   243. A method according to any of the preceding embodiments, wherein    the monomeric IL-10 is an Epstein Barr virus (EBV) IL-10 homolog of    SEQ ID No.: 3.-   244. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an amino acid substitution at position 75 of    SEQ ID No.: 3.-   245. A method according to any of the preceding embodiments, wherein    the EBV IL-10 includes an V31L amino acid substitution.-   246. A method according to any of the preceding embodiments, wherein    the VH region of the first antibody is from an anti-HIV monoclonal    antibody and the VL region of the second antibody is from an    anti-ebola monoclonal antibody.-   247. A method according to any of the preceding embodiments, wherein    the first peptide chain is an amino acid sequence selected from SEQ    ID Nos.: 26, 41, or 48.-   248. A method according to any of the preceding embodiments, wherein    the second peptide chain is an amino acid sequence selected from SEQ    ID Nos.: 27, 42, 49.-   249. A method according to any of the preceding embodiments, further    comprising a linker between the VH regions and the VL regions.-   250. A method according to any of the preceding embodiments, wherein    VH and VL each comprise one or more amino acid substitutions that    reduce antigenicity in the patient.-   251. A method according to any of the preceding embodiments, wherein    the first antibody is from an anti-HIV monoclonal antibody and the    second antibody is from an anti-ebola monoclonal antibody.-   252. A fusion protein of formula (I-VII)    -   IL10-L¹-X¹-L¹-X²-L¹-IL10 (Formula I); (Z)_(n)—X¹-L²-Y²-L¹-IL10        (Formula II); IL10-L¹-Y¹-L²-X²—(Z)_(n) (Formula III);        X¹-L²-X²-L¹-IL10 (Formula IV); IL10-L¹-X¹-L²-X² (Formula V);        X¹-L¹-IL10 (Formula VI); IL10-L¹-X2 (Formula VII); or any        combination thereof,        wherein    -   “IL-10” is a monomer sequence selected from SEQ ID Nos: 1, 3,        14, 18, 15, 19, 16 20, 55, 57, or 59; more preferably the        “IL-10” consists of SEQ ID No: 55, 57, or 59;    -   “L¹” is a linker of SEQ ID No: 31 or 54;    -   “L²” is a linker of SEQ ID No: 30;    -   “X¹” is a VH region obtained from a first antibody specific for        epidermal growth factor receptor (EGFR); CD52; various immune        check point targets, such as but not limited to PD-L1, PD-1,        TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2; HER2; EpCAM; ICAM        (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TFGβ        Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin        SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1;        SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola;    -   “X₂” is a VL region obtained from the same antibody as X₁;    -   “Y₁” is VH region obtained from a second antibody specific for        epidermal growth factor receptor (EGFR); CD52; various immune        check point targets, such as but not limited to PD-L1, PD-1,        TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2; HER2; EpCAM; ICAM        (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TFGβ        Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin        SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1;        SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola;    -   “Y₂” is a VL region obtained from the same antibody as Y₁;    -   wherein X and Y are obtained from the same or different        antibody;    -   “Z” is a cytokine selected from IL-10, IL-10 variant molecule        IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15,        IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β,        -γ, TGF-β, or tumor necrosis factors -α, -β, basic FGF, EGF,        PDGF, IL-4, IL-11, or IL-13;    -   “n” is an integer selected from 0-2.-   253. The fusion protein according to the preceding embodiment,    wherein formula II and III are capable of forming a fusion protein    complex where the IL-10 monomer from each of formula II and III are    capable of forming a functional homodimeric IL-10 or variant    thereof.-   254. The fusion protein according to any of the preceding    embodiments, wherein formula II is SEQ ID Nos: 24, 26, 28, 41, 48,    or 50.-   255. The fusion protein according to any of the preceding    embodiments, wherein formula III is SEQ ID Nos: 25, 27, 29, 42, 49,    or 51.-   256. The fusion protein according to any of the preceding    embodiments, wherein the fusion protein complex is formed between    SEQ ID Nos: 24 and 25; 26 and 27, 28 and 29; 41 and 42; 48 and 49;    or 50 and 51.-   257. The fusion protein according to any of the preceding    embodiments, wherein formula IV and V are capable of forming a    fusion protein complex where the IL-10 monomer from each of formula    IV and V are capable of forming a functional homodimeric IL-10 or    variant thereof.-   258. The fusion protein according to any of the preceding    embodiments, wherein formula IV is SEQ ID Nos: 35, 38, 46, 48, or    50.-   259. The fusion protein according to any of the preceding    embodiments, wherein formula V is SEQ ID Nos: 36, 39, 47, 49, or 51.-   260. The fusion protein according to any of the preceding    embodiments, wherein the fusion protein complex is formed between    SEQ ID Nos: 35 and 36; 38 and 39, 46 and 47; 48 and 49; or 50 and    51.-   261. The fusion protein according to any of the preceding    embodiments, wherein formula VI and VII are capable of forming a    fusion protein complex where the IL-10 monomer from each of formula    VI and VII are capable of forming a functional homodimeric IL-10 or    variant thereof.-   262. The fusion protein according to any of the preceding    embodiments, wherein formula I is SEQ ID Nos: 33-34, 40, 43-44, 45,    52 53, 61, 63, 65, or 67.-   263. The fusion protein according to any of the preceding    embodiments, wherein “n”≥1 and Z is IL-2, 11-7, IL-12, IL-15 or any    combination thereof.-   264. The fusion protein according to any of the preceding    embodiments, wherein Z is conjugated onto the N-terminal end of X¹,    Y¹, or both.-   265. A method of treating cancer comprising administering to a    patient in need thereof a composition comprising a fusion protein    according to according to any of the preceding embodiments.-   266. The method according to any of the preceding embodiments,    wherein the fusion protein is SEQ ID Nos: 28-29, 35-36, 38-39,    46-47, 52 53, 61, 63, 65, or 67.-   267. The method according to any of the preceding embodiments,    wherein the fusion protein forms a protein complex and the protein    complex is formed between SEQ ID Nos: 28 and 29; 35 and 36; 38 and    39; or 46 and 47.-   268. The method according to any of the preceding embodiments,    wherein the composition comprises a fusion protein of SEQ ID Nos:    33, 34, 44, 52 or 53, 61, 63, 65, or 67.-   269. The method according to any of the preceding embodiments,    wherein the fusion protein comprises an IL-10 consisting of DV07 of    SEQ ID No. 59.-   270. A method of treating inflammatory disease comprising    administering to a patient in need thereof a composition comprising    a fusion protein according to any of the preceding embodiments.-   271. The method according to any of the preceding embodiments,    wherein the fusion protein is SEQ ID Nos: 26-27, 41-42, 48, or 49.-   272. The method according to any of the preceding embodiments,    wherein the fusion protein forms a protein complex and the protein    complex is formed between SEQ ID Nos: 26 and 27; 41 and 42; 48 and    49.-   273. The method according to any of the preceding embodiments,    wherein the composition comprises a fusion protein of SEQ ID Nos:    37, 40, or 43.-   274. The method according to claim 18, wherein the fusion protein    comprises an IL-10 consisting of DV06 of SEQ ID No. 57.-   275. A method of treating a lipid based disease comprising    administering to a patient in need thereof a composition comprising    a fusion protein according to any of the preceding embodiments.-   276. The method according to any of the preceding embodiments,    wherein the fusion protein is SEQ ID Nos: 24-25, 50 or 51.-   277. The method according to any of the preceding embodiments,    wherein the fusion protein forms a protein complex and the protein    complex is formed between SEQ ID Nos: 24 and 25; and 50 and 51.-   278. The method according to any of the preceding embodiments,    wherein the composition comprises a fusion protein of SEQ ID No: 45.

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1. A fusion protein of formula (I, IV, V, VI, or VII)IL10-L¹-X¹-L¹-X²-L¹-IL10   (Formula I);X¹-L²-X²-L¹-IL10   (Formula IV);IL10-L¹-X¹-L²-X²   (Formula V);X¹-L¹-IL10   (Formula VI);IL10-L¹-X2   (Formula VII), wherein “IL-10” is a monomer sequenceselected from SEQ ID Nos: 3, 15, 19, or 51; “L¹” is a linker of SEQ IDNo: 31 or 54; “L²” is a linker of SEQ ID No: 30; “X¹” is a VH regionobtained from a first antibody specific for epidermal growth factorreceptor (EGFR); CD52; various immune check point targets, such as butnot limited to PD-L1, PD-1, TIM3, BTLA, LAG3 or CTLA4; CD20; CD47;GD-2;HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2;EDB-FN; TFGβ Trap; MadCam, β7 integrin subunit; α4β7 integrin; α4integrin SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1;SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV, or Ebola; “X²” is aVL region obtained from the same antibody as X¹.
 2. (canceled) 3.(canceled)
 4. (canceled)
 5. (canceled)
 6. The fusion protein accordingto claim 1, wherein formula IV and V are capable of forming a fusionprotein complex where the IL-10 monomer from each of formula IV and Vare capable of forming a functional homodimeric IL-10 or variantthereof.
 7. The fusion protein according to claim 6, wherein formula IVis SEQ ID No:
 48. 8. The fusion protein according to claim 6, whereinformula V is SEQ ID No:
 49. 9. The fusion protein according to claim 6,wherein the fusion protein complex is formed between SEQ ID Nos: 48 and49.
 10. The fusion protein according to claim 1, wherein formula VI andVII are capable of forming a fusion protein complex where the IL-10monomer from each of formula VI and VII are capable of forming afunctional homodimeric IL-10 or variant thereof.
 11. The fusion proteinaccording to claim 1, wherein formula I is SEQ ID Nos: 37, 40, 43.12-26. (canceled)
 27. The fusion protein according to claim 1, whereinthe VH region and the VL region are from an anti-Ebola antibody; andwherein the VH region and VL region comprise 6 CDR regions engraftedwith 6 CDR regions from an antibody selected from EGFR; CD52; PD-L1;PD-1; TIM3; BTLA; LAG3; CTLA4; CD20; CD47;GD-2; HER2; EpCAM; ICAM-1;ICAM-2; ICAM-3; ICAM-4; ICAM-5; VCAM, FAPα; 5T4; Trop2; EDB-FN; TFGβTrap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin SR-A1;SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2;SR-G; SR-H1; SR-H2; SR-I1; or SR-J1.
 28. The fusion protein according toclaim 27, wherein the VH region and the VL region from the anti-Ebolaantibody is engrafted with 6 CDR regions from an anti-MadCam antibody.29. The fusion protein according to claim 1, wherein the VH region andthe VL region comprise modifications that reduce the antigenicity in asubject.
 30. A pharmaceutical composition comprising a therapeuticallyeffective amount of the fusion protein according to claim 1, and apharmaceutically acceptable carrier, excipient, preservative,stabilizers, or any combination thereof.